Sequence-specific in vivo cell targeting

ABSTRACT

Enhanced, specific nucleic acid targeting complexes comprising endo and exonuclease activity, and related methods that allow both targeted degradation of specific and/or non-specific nucleic acids in vivo and specific temporal regulation of nuclease activity to prevent off-target activity are disclosed herein. Through practice of the disclosure, nucleic acids, and cells harboring them, such as cancer cells or pathogens, are selectively degraded in vivo.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No. 17/033,313, filed Sep. 25, 2020, which is a continuation of International Patent Application No. PCT/IB2019/000346, filed Mar. 27, 2019, which claims the benefit of U.S. Provisional Application No. 62/652,150 filed Apr. 3, 2018, U.S. Provisional Application No. 62/724,199 filed Aug. 29, 2018, and Korean patent application No. KR 10-2018-0035298, filed on Mar. 27, 2018, all of which are incorporated herein by reference in their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 9, 2022, is named 53470-706_302SL.txt and is 965,054 bytes in size.

BACKGROUND

Targeting specific cells for degradation or cell death remains a challenge. Cancer or pathogen cell populations are often difficult to target without also impacting undiseased host cell populations. Consequently, particularly for weakened individuals but also for individuals suffering from or identified as having a condition in general, treatment regimens convey negative health consequences that may be harmful or that may negatively impact the efficacy of the treatment regimen.

SUMMARY OF THE INVENTION

Provided herein are embodiments related to nucleic acid targeting to improve health, ameliorate at least one symptom of a disorder, reduce a pathogen load or to target particular cells such as cancer cells for selective elimination. Methods and compositions herein relate to the selective targeting of particular nucleic acid molecules for both endonucleolytic and exonucleolytic degradation. Target nucleic acids are identified through base pairing by a guide nucleic acid molecule, such as a guide nucleic acid molecule in a CRISPR or other nucleoprotein complex. Targeted nucleic acids are cleaved so as to introduce a double-strand break. Target nucleic acid degradation is then facilitated through exonucleolytic activity, such as exonucleolytic activity associated with or fused to the protein component of the nucleoprotein complex, which causes the nucleic acid to be degraded selectively from an endonucleolytically introduced cleavage site. In addition, nonspecific exonuclease activity of CRISPR or other nucleoprotein complex is induced by crRNA-guided sequence specific endonuclease activity, which degrade cellular DNA/RNA molecules non-selectively.

Further summary is obtained in part through reference to the claims as listed below.

In various aspects, the present disclosure provides a method of selectively inducing cell death in a cell, the method comprising: a) administering to a subject in need thereof, a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity a guide nucleic acid comprising a sequence complementary to a target nucleic acid in the cell; and b) cleaving the target nucleic acid, thereby inducing cell death.

In some aspects, the target nucleic acid is associated with a disorder. In some aspects, the target nucleic acid is in a cancer cell of the subject. In some aspects, the target nucleic acid is absent in a healthy cell of the subject. In some aspects, the target nucleic acid comprises a sequence associated with a cancer. In some aspects, the target nucleic acid comprises RNA.

In other aspects, the target nucleic acid comprises DNA. In some aspects, the target nucleic acid is within a region comprising a chromosomal abnormality. In some aspects, the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, copy number variations, an indel, and an isochromosome. In some aspects, the cell is in a plurality of cells.

In further aspects, the plurality of cells comprises a healthy cell. In some aspects, after the administering, the healthy cell lives. In some aspects, after the administering, the healthy cell proliferates. In some aspects, the cleaving comprises cleaving at one or more than one cleavage site in the cell. In some aspects, the target nucleic acid is associated with a disorder. In some aspects, the cancer comprises lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, or cervical cancer.

In some aspects, the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer, a translocation, or a sequence associated with cancer progression. In some aspects, the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, HER2, KIF5A, IRX1, ADAMTS16, GNPDA2, KCNE2, SLC15A5, SMIM11, DACH2, HERC2P2, CD68SHBG, ERBB2, KRT16, LINC00536, TRPS1, CDK8, TRAPPC9, HERC2P2, SIRPB1, MRC1, ATP11A, POTEB, HERC2P2, PRDM9, CDKN2B, HPV, LINE2 (MT2), CCR5, or HPRT1.

In some aspects, the chimeric polypeptide further comprises inducible non-specific nuclease activity. In some aspects, the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. In some aspects, the first domain comprises a Cas12a (or Cpf1) domain, a Cas12a domain, a Cas12b domain, a Cas12c domain, a Cas12d domain, a Cas12e domain, a Cas12f domain, a Cas12g domain, a Cas12h domain, a Cas12i domain, a Cas13a domain, a Cas13b domain, a Cas14 domain, or a Cas9 domain. In further aspects, the first domain comprises a Cas12a (or Cpf1) domain. In other aspects, the first domain comprises a Cas13a or Cas13b domain. In still other aspects, the first domain comprises a Cas9 domain. In some aspects, the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305].

In some aspects, the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. In some aspects, the chimeric polypeptide generates a 3′ OH overhang. In some aspects, the chimeric polypeptide exposes a recessed 3′ OH. In some aspects, the second domain comprises an enzyme having cleaved end resection activity. In some aspects, the second domain comprises mung bean nuclease. In some aspects, the target nucleic acid is absent in the healthy cell of the subject.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including a Korean patent application, KR 10-2018-0035298, filed on Mar. 27, 2018, which is incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Some understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows a schematic of CRISPR/Cas complex single and double-stranded nucleic acid endonuclease and exonuclease activity.

FIG. 2 shows a schematic of CRISPER/Cas activity on target and nontarget nucleic acid substrates.

FIG. 3 depicts in vitro nuclease activity assay results.

FIG. 4 shows in vivo toxicity assay results.

FIG. 5 shows a PX459 plasmid vector system for SpCas9 and sgRNA transfection with puromycin resistance. The PX459 plasmid vector map is from www.addgene.org/62988. SpCas9 and sgRNA is expressed after cleavage of BbsI enzyme and following insertion of target sequence dsDNA oligo.

FIGS. 6A-6B show Cas9 guide RNA derived multi-cleavage induction and cell death in an EGFR mutant lung cancer cell line, HCC827, six days after neon electroporation of PX459 plasmid DNA targeting copy number variation. FIG. 6A shows images of the cells, which from left to right are the following treatments: electroporation pulse only without any vectors (control), electroporated with vectors that target the wild-type EGFR sequence (control), and electroporated with vectors that target the mutant sequences. FIG. 6B shows a bar graph of the number of cells after each of the above mentioned treatments.

FIG. 7 shows microscopic data confirming cell morphology for each sample in FIG. 8. In treatments targeting the NT1, CCR5, and HPRT1 loci, many cells survived. In treatments targeting the MT2, SMIM11, GNPDA2, SLC15A5, and KCNE2 loci, most cells were dead.

FIG. 8 shows a graph of cell viability in each treatment group, including treatments targeting NT1, CCR5, HPRT1, MT2, SMIM11, GNPDA2, SLC15A5, and KCNE2 loci.

FIG. 9 shows data of cell viability after transfection of CRISPR/Cas9 constructs targeting different gene loci including lipo (lipofectamine only), NT1, CCR5, HPRT1, MT2, GNPDA2, SLC15A5, and KCNE2.

FIG. 10 shows a bright field image and fluorescence microscopy images of CRISPR/Cas9 constructs targeting NT1 (negative control, no matched sequence in the human genome) and GNPDA2.

FIG. 11 shows data of cell viability after transfection of CRISPR/Cas9 constructs targeting different gene loci including CCR5, HPRT1, MT2, IRX1, and ADAMTS16.

FIGS. 12A-12B show data of cell viability after transfection of CRISPR/Cas9 constructs targeting different gene loci.

FIG. 12A shows data of cell viability after transfection of cells with CRISPR/Cas9 constructs targeting different gene loci including pulse only, NT1, HPRT1, CCR5, and MT2 at 24 h, 42 h, and 72 h post-transfection using a cell titer glo assay.

FIG. 12B shows data of cell viability after transfection of cells with CRISPR/Cas9 constructs targeting different gene loci including pulse only, NT1, HPRT1, CCR5, and MT2 by cell counting.

FIG. 13 shows bright field images of cells after transfection of CRISPR/Cas9 constructs targeting different gene loci including pulse only, NT1, HPRT1, CCR5, and MT2, with arrows point to live cell colonies.

FIG. 14 shows data of cell viability after transfection of cells with CRISPR/Cas9 constructs targeting different gene loci including pulse only, pET21a, MT2, CD68, DACH2, HERC2P2, SHBG, and no pulse at 24 hr, 44 hr, and 51 hr post-electroporation.

FIGS. 15A-15C show the duplicated region of ERBB2 in SK-BR-3 with whole genome sequence data.

FIG. 15A shows the SK-BR-3 ERBB2 region WGS data from Nattestad et al.

FIG. 15B shows the SK-BR-3 ERBB2 CNV data from Nattestad et al.

FIG. 15C shows the SK-BR-3 ERBB2 CNV junction sequence, which was inferred from Nattestad et al. Figure discloses SEQ ID NOS 3292-3298, respectively, in order of appearance.

FIGS. 16A-16B show data from electroporation of cells with CRISPR/Cas9 constructs targeting different gene loci.

FIG. 16A shows data from electroporation of cells with CRISPR/Cas9 constructs targeting different gene loci. Samples include pulse only, NT1, MT2, ERBB2, KRT16, and no pulse.

FIG. 16B shows the raw data associated with FIG. 16A.

FIG. 17 shows data from electroporation of cells with CRISPR/Cas9 constructs targeting different gene loci. Samples include pulse only, NT1, HPRT1, MT2, ERBB2, and KRT16.

FIGS. 18A-18C show HeLa duplicated sequences.

FIG. 18A shows HPV and HeLa cell integration data from Adey, A. et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500, 207-211 (2013).

FIG. 18B shows HPV sequence data in HeLa cell inferred from Meissner, J. D. Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J. Gen. Virol. 80, 1725-1733 (1999), Landry, J. J. M. et al. The genomic and transcriptomic landscape of a HeLa cell line. G3:Genes|Genomes|Genetics 3, 1213-24 (2013), Adey, A. et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500, 207-211 (2013), and Liu, Y., Lu, Z., Xu, R. & Ke, Y. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology. Oncotarget 7, 5852-5864 (2016).

FIG. 18C shows an electrophoresis gel picture of PCR product which are repeat region between HPV sequence in HeLa cell, based on Meissner et al, Landry et al, Adey et al, and Liu et al.

FIGS. 19A-19B show HeLa cell death after targeting HeLa CNV or HPV gene.

FIG. 19A shows the live cell number after electroporation of cells with CRIPSR/Cas9 targeting CCR5, MT2, and PRDM9.

FIG. 19B shows the live cell number after electroporation of cells with CRIPSR/Cas9 targeting CCR5, MT2, PRDM9, and HPV_1.

FIGS. 20A-20B show HeLa cell death after targeting HeLa CNV or HPV gene.

FIG. 20A shows a graph of cell viability after electroporation of cells with CRIPSR/Cas9. Samples include pulse only (GFP), CCR5, MT2, HPV_1, and PRDM9, at 24 h, 48 h, and 72 h post-transfection.

FIG. 20B shows a graph of HeLa cell death by targeting the HPV gene using a cell titer glo assay, with samples of NT1, NT2, NT3, CCR5, MT2, and HPV_1.

FIG. 21 shows cell viability after transfection of HT29 cells with CRIPSR/Cas9 targeting various gene loci. Samples include NT1, CCR5, HPRT1, MT2, TRAPPC, LINC00536, TRPS1, and CDK8.

FIG. 22 shows cell viability after transfection of H1563 cells with CRIPSR/Cas9 targeting various gene loci. Samples include CCR5, SMIM11, GNPDA2, SLC15A5, and KCNE2.

FIG. 23 shows microscopic images of cell viability after transfection of cells with CRIPSR/Cas9 targeting various gene loci.

FIG. 24 shows cell viability via AnnexinV quantification after transfection of cells with CRIPSR/Cas9 targeting various gene loci.

FIG. 25 shows, at top, images of cells after transfection of cells with CRISPR/Cas9 or CRISPR PLUS™ systems targeting various gene loci. At bottom is a graph of cell viability of each treatment.

FIG. 26 shows RNP neon electroporation showing Cas9 guide RNA derived multi-cleavage induction and cell death in an EGFR mutant lung cancer cell line, HCC827.

FIG. 27 shows RNP lipofection of CRISPR/Cas9 or CRISPR PLUS™ systems 2 days after lipofection, wherein the Cas9 guide RNA induces multi-cleavage and cell death in an adenocarcinoma cell line, H1563.

FIG. 28 shows, at top, images of cells after electroporation of cells with CRISPR/Cas9 or CRISPR PLUS™ systems targeting various gene loci. The bottom graphs show cell viability after electroproation in H1299 cells, an adenocarcinoma cell line.

FIG. 29 shows cell viability, as measured by a Cell Titer Glo assay, after targeting CNVs in HT-29 cells.

FIG. 30 shows the target sequences for AsCpf1_crRNA and the Cpf1 crRNA binding site WT. Figure discloses SEQ ID NO: 3299.

FIG. 31 shows sequence specific cancer cell killing effect by Cas12a and single or multi-target crRNAs, including images of cells at top and a bar graph of the live cells in each treatment group at bottom.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods related to the selective targeting of nucleic acids, such as nucleic acids in pathogen cells or in cells having dysregulated growth or proliferation often associated with cancer. Through the disclosure herein, targeted nucleic acids are selectively degraded, so as to often lead to their clearance from an individual. Cellular DNA/RNA also are degraded by nonspecific exonuclease activity. Clearance is often effected through cell death such that cells harboring the nucleic acid are selectively killed. However, extracellular nucleic acids such as viral nucleic acids are also targeted through some embodiments of the disclosure herein.

Targeted clearance is accomplished through a combination of sequence-specific nucleic acid detection and an endonucleolytic/exonucleolytic degradation activity. Compositions and methods often comprise a sequence specificity component. In many embodiments herein, the moiety comprises a guide nucleic acid such as a guide RNA or guide DNA that achieves sequence specificity through base pairing to a reverse-complementary (often referred to as merely ‘complementary’) base sequence in a target nucleic acid. However, alternatives such as TALEN, zinc finger or other programmable, specific sequence-identifying moiety are also contemplated. In exemplary embodiments, sequence specificity is conveyed through a guide RNA that is reverse complementary to a segment of a target nucleic acid molecule.

Upon identifying a target nucleic acid, clearance is effected through combined endonuclease/exonuclease activity, such as that conveyed through a protein component of a nucleoprotein complex such as a CRISPR/Cas nucleoprotein complex. Further, non-specific exonuclease activity of the protein component of a nucleoprotein complex is also activated upon identifying a target nucleic acid. The non-specific exonuclease activity degrades non-specifically cellular DNA/RNA molecules, thereby causing cell death. Exonuclease activity herein means that enzymes work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. Endonucleolytic cleavage is effected through CRISPR/Cas endonucleolytic activity or through other endonucleolytic activity associated with or directed by the sequence-specifying moiety as discussed above. Such activity cleaves the target nucleic acid at, near, or in a manner directed by the sequence specifying moiety, such that nucleic acids harboring a target sequence are selectively cleaved. Endonucleolytic cleavage herein means cleavage of the phosphodiester bond within a polynucleotide chain by enzymes.

Degradation is further facilitated by a second nucleic acid degrading activity. This activity is in some cases sequence-specific or otherwise directed by the sequence specifying moiety. Alternately, the activity is targeted to double-strand breaks, independent of sequence, but such that double-strand or single-strand breaks generated by the endonucleolytic activity above will be selectively or nonselectively targeted for further degradation.

In exemplary complexes and methods herein, the second activity is an exonuclease activity and is delivered in association with the endonucleolytic activity, such as through fusion to the endonucleolytic activity in a chimeric polypeptide having a common phosphodiester backbone, such that the endonucleolytic activity is directed to the endonucleolytic cleavage site. In another implementation, co-administration or other mechanism for delivery of the second activity is also contemplated. Nucleic acids that are specifically identified, specifically cleaved endonucleolytically, and further subjected to exonuclease activity or other activity that ‘proliferates’ a DNA lesion directed by the sequence specificity moiety are very often damaged such that they no longer encode sufficient information to encode ongoing cellular or viral activity. That is, upon being subjected to a composition or method as disclosed herein, a nucleic acid is likely to have lost sufficient sequence such that it no longer encodes at least one protein necessary for cellular function, thereby killing the cell. Similarly, nucleic acids subjected to a composition or method as disclosed herein are likely to be identified as damaged by a host cell independent of how much of their coding capacity is lost through endo- and exonucleolytic activity, such that the cell is targeted for degradation through internal DNA integrity checkpoints leading to cell apoptotic mechanisms. That is, non-specific exonuclease activity induced by activation of crRNA-guided sequence-specific nuclease activity degrades non-specifically cellular DNA/RNA molecules, which causes a fatal effect in the cell.

Temporal regulation is achieved through temporal, specific inactivation of nuclease complexes by at least one of enzymatic inactivation and sequestration via antibody binding. Temporal inactivation conveys the benefit of clearing the nuclease from an individual, either locally or systemically, so as to reduce the risk of off-target activity, particularly in situations where targeted nucleic acids are largely or completely cleared from an individual or a particular site at an individual.

As discussed above, some compositions and methods comprise temporal regulation of activity through administration of a treatment or composition that impacts at least one of nucleic acid recognition, endonucleolytic activity and exonucleolytic activity or other activity consistent with the disclosure herein. An individual is subjected to a method or administered a composition consistent with the disclosure herein and is administered a follow-on composition or treatment so as to inactivate the nuclease-activity composition. Examples include but are not limited to local heat administration, zn^(2+ion) or its variants, for example, ZnSO₄, or any others shown in Table 4 or their variants, administration or other composition administration that inactivates, destabilizes or otherwise neutralizes the nuclease activity, template-killing nucleic acids that stably bind the guide nucleic acid, rendering it unable to direct further degradation, endonuclease-killing nucleic acids that stably bind the CRISPR/Cas complex, rendering it unable to processively degrade nucleic acid substrate in a target-directed manner, and antibodies that bind and impact, up to and including inactivate, a complex responsible for specific nucleic acid identification or targeted degradation. Accordingly, through practice of the disclosure, herein, one accomplishes specific targeting and degradation of particular nucleic acids, both through endonucleolytic cleavage and through proliferation of a cleavage event such that a substantial or in some cases catastrophic lesion is introduced in a nucleic acid molecule, up to and including deletion of an entire nucleic acid am or an entire nucleic acid molecule. Similarly, one accomplishes specific control of this activity both through selection of a guide RNA or other programmable sequence specifying moiety so as to direct nucleic acid degrading activity to a particular target or class of target, and in some cases through temporal termination of activity so as to clear the activity from an individual once a particular time period has passed.

Context of the disclosure. Cancer is a major human health issue. One of the most prominent properties of cancer is indefinite proliferation, which has been used to select and develop anticancer drugs that target this trait. Therapies targeting highly proliferative cells in the body are often used as a first line chemotherapy or radio therapy. These methods, however, also target normal cells that highly proliferate in the body, such as hair and immune cell populations, which may result in moderate to severe side effects and sufferings from the patients. The development of highly specific anti-cancer therapeutics that selectively target the cancer cells in the body provides the potential for substantial benefits to patients.

Recently, immuno-oncology using a patient's immune system for cancer treatment has achieved substantial progress in anti-cancer therapies. CAR-T cell therapy, for example, uses patient-derived immune T cells that express antigen receptors in the cell membrane through genetic manipulation or other approaches. The antigen receptors recognize specific membrane proteins in cancer cells and destroy them. However, the strength of interaction of biomolecules between cancer cells and anti-cancer drugs is often not satisfactory for complete specificity, often resulting in off-target effects or incomplete targeting. Thus, even though targeted cancer therapy and immuno-oncology are considered to be less toxic than traditional cancer therapy, they still have substantial side effect antigen-directed therapies often exhibit both side effects and remission followed by relapse of cancer cell growth and progression. Also, finding specific membrane proteins in cancer cells remains challenging.

Existing Approaches. Targeted cancer therapies show promise and have been actively developed. Targeted cancer therapies are anti-cancer therapeutics that block cancer growth by inhibiting specific molecular targets or pathways that are involved in the cancer pathogenesis. Targeted cancer therapies are sometimes called “molecularly targeted drugs” or “molecularly targeted therapies”. Targeted therapies are currently the focus of much anticancer drug development.

Targeted approaches require targets specific to one or another cell type to be eliminated. Many approaches identify specific targets by comparing the amounts of total proteins in cancer cells with those in normal cells. Proteins that are specifically present in cancer cells or that are more abundant in cancer cells are potential targets. Stronger candidates are known to be important for the cancer pathogenesis. A well-known example of differentially expressed target protein is the human epidermal growth factor receptor 2 protein (HER-2). HER-2 is expressed at high levels on the surface of some cancer cells. Several targeted therapies have been developed against HER-2 including trastuzumab (Herceptin®), which is approved to treat certain breast and stomach cancers that overexpress HER-2.

Another approach is to detect mutant proteins that cause cancer progression. For example, the cell growth signaling protein BRCA1 or BRAF is present in an altered form in many breast cancer and melanomas, respectively. Many targeted therapeutics are developed to target these mutant forms and are approved to treat patients with inoperable or metastatic cancers that contain these altered proteins.

Abnormalities in chromosome specifically present in cancer cells are also used for potential targets. These chromosome abnormalities often create gene fusion, resulting in the expression of fusion protein that play pivotal role in cancer progression. Such abnormal fusion proteins specifically present in cancer cells can be used for the development of targeted therapies.

The imatinib mesylate (Gleevec) targets the BCR-ABL fusion protein that has been known to be formed by fusion of two genes and exist in some leukemia cells and drives the leukemia progression.

Most targeted therapies use either small molecules or monoclonal antibodies to block or neutralize specific proteins or pathways present specifically in cancer cells. Small molecule candidates are usually identified through high-throughput screens, in which the effects of test compounds on a specific target protein are massively screened. For the production of monoclonal antibodies, specific target proteins that are specifically present in cancer cells are used to inject animals (usually mice) to induce the animals to make antibodies against the target. These antibodies are then tested to find those that bind best to the target without binding to non-target proteins.

Spatiotemporally specific nucleic acid based approaches. Cancer cells often exhibit increased mutation rates relative to healthy cells. These increased mutation rates may manifest themselves in at least one of chromosomal variations, such as insertions, deletions, duplications, transversions or translocations, or in local mutations such as single- or few-base insertions, deletions, or single nucleotide polymorphisms (SNPs). These mutations are not always causative or even specifically indicative of actively dividing or metastatic cells within a tumor cell population. However, they often identify cells as including genetic mutation on specific genomic sites that do not exist in normal cells, depending on the originated tissues and the type of cancer. Those cancer-specific mutation such as SNPs are used to cancer cell specific markers in approaches disclosed herein.

CRISPR/Cas proteins are nucleic acid-guided enzymes (e.g., RNA guided) that recognize specific sequence of the DNA or RNA, and cleave them as a component of bacterial adaptive immune system. These specific DNA/RNA cleavage systems have been considered as attractive sequence-specific genome editing tools in vivo. For example, CRISPR/Cas9-based genome editing (GE) tools show promise in the field of disease therapeutics and agricultural traits. Genome editing of CRISPR/Cas-gRNA complex is achieved by site-specific dsDNA/RNA cleavage by RNA-guided endonuclease proteins such as Cas12a (or Cpf1), a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas13a, a Cas13b, a Cas14, or a Cas9, or others, followed by sequent cellular repair mechanism. A number of different programmable endonucleases, such as Cas endonucleases, that cleave nucleic acids are consistent with the present disclosure. Nucleic acid cleaving programmable Cas endonucleases are described, for example, in Harrington et al. (Science. 2018 Nov. 16; 362(6416):839-842. doi: 10.1126/science.aav4294. Epub 2018 Oct. 18.), which whole reference is hereby incorporated in its entirety. Nucleic acid cleaving programmable Cas endonucleases are described, for example, in Yan et al. (Science. 2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271. Epub 2018 Dec. 6.), which whole reference is hereby incorporated in its entirety. Other references, known to those of skill in the art, provide a broad range of Cas proteins, all of which are consistent with the present disclosure, including any of Cas12a (or Cpf1), a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas13a, a Cas13b, a Cas14, or a Cas9. Proteins that bind to and are guided by a guide RNA to direct sequence specific cleavage or that, otherwise, bind nucleic acid sequences in a sequence specific way to trigger non-specific cleavage are consistent with the present disclosure. Guide RNA that has a complementary sequence to the target DNA/RNA confer the sequence specificity of CRISPR/Cas-gRNA complex-dependent target site cleavage.

The genome editing function of members of CRISPR/Cas proteins, CRISPR/Cas12 and CRISPR/Cas13a is activated by gRNA-guided DNA/RNA binding, which subsequently activates non-specific nuclease activity that degrade single strand DNA or RNA molecules (FIG. 1). Site specific dsDNA/RNA cleavage and non-specific single strand DNA/RNA cleavage of CRISPR/Cas12 and CRISPR/Cas13a are separate activities.

Enhancement of this exonuclease activity, such as through co-administration or chimeric fusion of exonuclease activity targeting both double stranded and single stranded DNA and RNA that is activated by gRNA-guided target site binding to a CRISPR complex increases nucleic acid damage and can induce cell death by destroying essential cellular DNA and RNA molecules nonspecifically and irreversibly.

Accordingly, sequence specific complexes such as CRISPR/Cas12 and CRISPR/Cas13a or other complexes referred to herein that specifically target cells such as cancer cells using selectively activated non-specific nuclease function by gRNA-guided binding to specific target sites present in cancer cells or other target cells allow one to specifically remove undesired or detrimental nucleic acids or cells that harbor them, while reducing or eliminating the negative impact on healthy proliferating cells.

The genome editing function of nucleic acid sequence directed CRISPR/Cas endonucleases such as CRISPR/Cas12 and CRISPR/Cas13a is activated by gRNA-guided DNA/RNA binding to specific target site, which subsequently activate inherent non-specific nuclease activity of the CRISPR/Cas proteins that degrade single strand DNA or RNA molecules. CRISPR/Cas proteins that have enhanced genome editing efficiency through chimeric fusion of additive nuclease functions have strong non-specific double strand (ds)/single strand (ss) DNA and RNA nuclease function activated by gRNA-guided target binding of CRISPR/Cas proteins. These systems facilitate nucleic acid targeting and degradation, such as in cancer cells, through specific cleavage of molecules having a target sequence, followed by nonspecific single or double stranded exonuclease activity. After the completion of the effect, one may administer a specific inhibitor and selective antibody against CRISPR/Cas proteins to remove the function of the complex remaining in the body to abolish the unwanted side effect of the proteins.

In an aspect of the present disclosure, cancer cell specific mutations such as SNPs are recognized by gRNA of CRISPR nuclease complexes which contain complementary sequence to the specific mutations such as SNPs. Sequence specific binding between mutations such as SNPs on genome of cancer cells and gRNA activate the genome editing function of CRISPR nuclease proteins resulting the target DNA/RNA breakage. This activation subsequently activates the inherent non-specific exonuclease function of CRISPR nuclease and/or CRISPR PLUS™ that destroys cellular ds and ss DNA/RNA molecules irreversibly in cancer cell, resulting in cell death (FIG. 2).

After completion of therapeutic effect CRISPR nuclease complexes, the CRISPR/Cas terminator, such as ZnSO₄ or others as indicated herein, and/or a specific antibody against CRISPR nuclease and/or CRISPR PLUS™ are administrated to terminate unwanted CRISPR activity remained in the body to minimize side effects or otherwise clear the complexes.

Accordingly, in an aspect of the present disclosure, one achieves unprecedented cancer cell-specific targeting in the body through specific activation of CRISPR/Cas nuclease function, as well as termination of the unwanted activity of the protein complex in vivo through specific inhibitor and selective antibody.

Advantages of the approaches disclosed herein. Practice of the disclosure herein has numerous advantages both in the context of cancer therapy and elsewhere. By way of example, the binding between nucleotides molecules (DNA:DNA or DNA:RNA) that have complementary sequence is relatively strong and highly specific as compared with interaction between other biomolecules such as ligand:receptor and antigen:antibody. The cancer cell specific therapeutic effect of the present disclosure herein, as one example implementation of the present disclosure, is based on high specificity of complementary DNA:RNA binding, and thus can be customized for the needs of individual patients or cell types. The nuclease activity of CRISPR PLUS™-gRNA complex may depend on the existence of specific mutations such as SNPs in the genome of the cell that is complementary to the gRNA sequence in the cell. Thus, the cell specific therapeutic effect of the present disclosure herein is greater than that of many other anti-cancer therapies currently developed and used.

By way of example, the cell targeting is specific and readily individualized. Cancers often have their own specific markers depending on the origin and cause of carcinogenesis. Different patients may have different types of cancer. Thus, the same therapy may exhibit different efficacy in different patients. In the therapeutic approaches disclosed herein, the cancer of each patient may be optionally preliminarily analyzed to identify specific SNPs which are used to identify specific markers of cancer or of particular cancer cells in each patient. Thus, each CRISPR PLUS™-gRNA complex or other complex used for this therapy may be crafted to be specific to the particular cancer genome in question. Therefore, variation among treatment efficacies caused by underlying variation across cancer cells between patients may be greatly reduced, as it is an individual's particular cancer cell line that can be targeted.

Further, target selection is not limited by prior cancer knowledge or by cancer mechanism. Through the practice of the present disclosure herein, any number of sequence targets may be identified. There need not be a mechanistic link between a particular mutation such as an SNP and cancer or other disease progression. Rather, any mutation such as an SNP identified as correlating with some or all of the cells in a tumor population may be targeted. As cancer cell populations are often characterized by decreased nucleic acid reproductive fidelity, this feature of the present disclosure vastly increases the opportunities for targeting cells for degradation.

Additionally, active complexes are readily cleared. This allows the sequence specificity of the disclosure to be complemented by temporal specificity, such that the treatment regimen is readily stopped or inhibited after a selected time period. This allows one to further reduce off-target effects that may occur through persistence of the composition in non-target cells in an individual.

Target range. Much of the present disclosure herein relates to cancer cell targeting as an exemplary application or implementation of the present technology. However, one readily understands that the scope and applicability of the present disclosure expands beyond tumors or particular cancer cells and encompasses additional cells or nucleic acids for which their clearance may lead to a therapeutic effect or to amelioration of at least one symptom of a condition.

Communicable diseases, for example, present many of the challenges addressed by the present disclosure herein and are similarly amenable to symptom amelioration up to and including full treatment. Many disease pathogens, for example, present variable or difficult to detect cell surface proteins, or reside within host cells so as to complicate their identification by an antibody-based approach. Similarly, small molecule treatments for many diseases involve substantial side effects, often impacting patient compliance or complicating palliative, prophylactic or preventative treatment regimens.

Pathogens causative of diseases such as malaria, sleeping sickness, or tuberculosis, for example are difficult to treat. Cells either do not present surfaces for antibody based or host-directed treatment, or rapidly change their cell surfaces so as to evade immune responses. Alternately, tuberculosis cells reproduce slowly enough that therapeutics targeting particular steps in cellular proliferation must be administered for a period so long as to jeopardize treatment compliance.

Through the practice of the present disclosure herein, cell-specific target nucleic acid sequences may be readily identified in a cellular or viral pathogen genome. Using these sequences, one readily may obtain specific targets that facilitate the sequence specific degradation of pathogen nucleic acids in a manner consistent with the present disclosure above, recited in the context of cancer but, as is apparent from the present disclosure, readily applicable to a number of disorders.

Accordingly, the present disclosure herein is consistent with targeting particular cancer cells, for example, lung cancer or pancreatic cancer, but also with targeting a broad range of eukaryotic, eubacterial or viral pathogens, such as Plasmodium cells, Trypanosoma cells, Toxoplasma cells, opportunistic yeast or other cells, Giardia cells, staphylococcus infections, E. coli, or viral pathogens such as HIV, influenza, HPV, or any number of other transmissible or genetic diseases.

The present disclosure provides compositions of guide RNA and Cas9 systems that can be used in a method of inducing cell death by cleaving at one or more positions in a cell. In particular embodiments, the cell is a diseased cell, such as a cancer cell, and is in a larger plurality of cells containing healthy cells and diseased (or cancer) cells. The compositions disclosed herein can be administered to the larger plurality of cells to induce cell death in only the cancer cells by targeting the guide RNA to mutant sequences present only in the cancer cells. Moreover, the guide RNA and Cas9 compositions disclosed herein target multiple sites for cutting, inducing multi-cleavage of target nucleic acids and efficient cell death.

Similarly, the present disclosure herein is consistent with ameliorating symptoms of a broad range of contagious disorders such as malaria, toxoplasmosis, sleeping sickness, Chagas disease, tuberculosis, E. coli infections, strep throat, influenza, the common cold, AIDS, and any number of other disorders having a nucleic acid as an integral part of a causative agent of the disorder.

Further, in an aspect of the present disclosure, unknown disorders or pathogens may be similarly addressed, so long as one or more target nucleic acid sequences are available. Thus, for example, an individual suffering from an unknown ailment and identified as having a detectable amount of an unknown organism identified only by, for example, whole sample untargeted sequencing, may nonetheless be targeted for elimination through design of a guide sequence nucleic acid complex or other sequence specific nuclease moiety so as to specifically target nucleic acids having a target sequence selected from the unknown organism's genome. The subject may be monitored for amelioration of symptoms up to and including full treatment in response to administration of a complex or practice of a method disclosed herein, so as to address the disorder without first definitively identifying the pathogen.

Having provided present disclosure relevant to the compositions and methods overall, also provided are details and variants relevant to particular aspects of the compositions and methods disclosed herein. It is understood that particular details below are generally applicable and combinable among one another to gain a broader understanding of the full scope of the various embodiments of the present disclosure herein, and that the present disclosure is sufficiently flexible to allow variations and alternative combinations without falling outside of the scope of the present disclosure herein.

Sequence specificity. As discussed above, in an aspect, embodiments of the present disclosure benefit from a sequence-specifying moiety so as to direct the nuclease activity of the present disclosure to particular targets to the exclusion of others. In exemplary embodiments, sequence specificity is conveyed through a guide nucleic acid such as a guide RNA or guide DNA. Such a molecule may be assembled into a CRISPR/Cas or other suitable complex to convey sequence specificity through base pairing to a reverse-complementary base sequence in a target nucleic acid.

However, as discussed above, a number of alternative moieties may be consistent with the present disclosure herein. Particular alternatives may include sequence-specific or sequence-recognizing proteins such as TALEN repeats, zinc finger proteins, transcription factor moieties such as helix-loop-helix or other protein structure. A beneficial feature of such moieties is that they exhibit sequence specificity in their substrate binding, such that they may direct nuclease activity complexes to particular target nucleic acid segments, and in some cases discerning among alternatives having substantial identity, in some instances differing by a single base or perhaps only a single epigenetic modification such as a base methylation. Preferably, but not exclusively these moieties are programmable, such that a target sequence can be identified and a specificity moiety subsequently selected having a nucleic acid or protein sequence that is selected to correspond to the target nucleic acid segment. Alternatively, a collection of nucleic acid binding moieties may be selected such that, rather than programming a particular structure to convey nucleic acid specificity, one may select from among a diversity of nucleic acid binding moieties, such as transcription factor nucleic acid moieties, so as to come to a moiety having nucleic acid specificity sufficient to discern between target and healthy nucleic acid segments. In particular, when a target sequence differs substantially from host or patient DNA such that the variation is greater than one or a few bases, there is substantial flexibility in specificity moiety selection. For example, in the cases when the complex is intended to target a pathogen genome or a reverse-transcribed viral genome or inserted viral genome rather than a cancer mutant cell type, specificity moieties are readily obtained from the target organism's own transcription factor collection. Accordingly, some embodiments herein may comprise endonuclease and exonuclease activities as disclosed elsewhere herein in combination with a nucleic acid specificity moiety comprising a polypeptide specificity factors rather than a guide nucleic acid.

In an aspect of the present disclosure, target nucleic acid size may vary according to the amount of information needed to convey specificity and the system through which nuclease activity is conveyed. In the case of CRISPR or CRISPR PLUS™ systems, presented as lead exemplary embodiments herein, a target nucleic acid size, and therefore minimum guide RNA or DNA size, is constrained both by the size needed to convey specificity and the size necessary for assembly into nucleoprotein complexes and to result in stable annealing. Target regions must be long enough to convey stable annealing but not so long as to lead to annealing to single-base mis-paired segments. By way of example, exemplary guide nucleic acids may have a specificity-determining region that is often 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30 nucleotides. Target regions for protein-directed sequence specificity moieties may exhibit more variation.

A number of target sequence types are consistent with the present disclosure herein, ranging from single nucleotide polymorphisms to junctions indicative of chromosomal rearrangement events to sequence targets comprising completely non-native nucleic acid sequence. By way of example, in an aspect of the present disclosure, sequence types identified by a sequence specificity moiety such as a guide RNA may include single base substitutions, single base deletions or insertions, multibase substitutions, insertions or deletions, often flanked by sequence segments that do not differ between target and non-target nucleic acids (such as wild-type or healthy sequence flanking a single base mutation in a cancer cell line). Alternately, some target sequences are selected to span a junction indicative of a chromosomal event such as a large deletion, duplication indicative of insertion of one sequence segment into another or adjacent to another on a chromosome, an inversion event, a translocation or other chromosomal rearrangement. Often, sequence on either side of the event may correspond to sequence in a healthy cell, but the positioning of the segments relative to one another may be indicative of a mutation such as a mutation relevant to a cancer or other health condition.

In an aspect of the present disclosure, by way of example, target sequences may be selected as correlating to disease. In the case of cancer treatments, target sequences may be selected as occurring in all or a substantial part of a tumor population. For example, target sequences may be selected to target actively proliferating or otherwise dangerous cells in a tumor population, or to target a tumor population as a whole or to target cells both within and outside of a particular tumor population. Target sequences may be in some cases mechanistically implicated in the disease (such as mutations implicated in misregulation of cell cycle checkpoints, for example, or mutations in known oncogenes such as p53, RB, E2F, CDK or Cyclin family members, or growth regulators such as members of the mTOR or PI3K signaling pathways). However, in an aspect of the present disclosure herein, one is not limited to targets mechanistically implicated in a disorder; rather, any sequence segment identified as correlating with or occurring specifically, predominantly, or substantially in a target cell population may be used as a target sequence. Given that many cancer or tumor cell populations exhibit defects in nucleic acid replication and subsequently accumulate mutations, the present disclosure allows a broad range of targets to be selected from. Consequently, in an aspect of the present disclosure, selection of target sequences may be greatly facilitated, as it is not limited by sequences corresponding to genes implicated in a particular disease.

By way of example, target sequences may be in some cases selected from previously determined sequences implicated in cancer or other disease. Published pathogen genomes, for example, are ready sources of target sequence, and may be selected without further verification or after verification confirming that they are not also present in the host genome. Similarly, published cancer genome information may often be relied upon for target sequence candidates.

An example of published information relevant to target sequence selection is provided in Table 1, below.

TABLE 1  SEQ SEQ ID Cancer ID Gene Cancer Normal cell NO: cell NO: Mutation RRCA1 Breast  608 1  608 11 p.Tyrl30 Exon 7 :CAAAGTATGGGCT CAAAGTATGG Phe ACAGAAACCGTGC GCTTCAGAAA CAAAAG CCGTGCCAAA AG BRCA1 1615 2 1615 12 p.Lys467 Exon 10 :TGGGAAAACCTAT :TGGGAAAACC non-sense CGGAAGAAGGCA TATCGGTAGA AGCCTCC AGGCAAGCCT CC BRCA1 3845 3 3845 13 p.Leul209Lle Exon 11 :GGGGCCAAGAAA- :GGGGCCAAG TTAGAGTCCTCAG AAAATTAGAG AAGAG TCCTCAGAAG AG BRCA1 4260 4 4260 14 P Exon 11 :ATGATGAAGAAA :ATGATGAAGA Gly1348Asn GAGGAACGGGCTT AAG-- GGAAGA GAACGGGCTT GGAAGA BRCA1 3657 5 3657 15 p Glu114Arg Exon 11 :CATCTCAGGTTTG :CATCTCAGGT TTCTGAGACACCT TTGTTCT- GATGACC AGACACCTGA TGACC BRCA2 7466 6 7466 16 p.Arg2494Thr Exon 15 :ATATACAGGATAT :ATATACAGGA GCGAATTAAGAAG TATGTGAATT AAACAAA AAGAAGAAAC AAA TP53 Gastric  125 7  125 17 p.Leu20Cys :TAGGAGGCCGAG :TAGGAGGCCG CTCTGTTGCTTCG AGCTCT- AACTCCA TTGCTTCGAAC TCCA MSH2 Colon  126 8  126 18 p.MetlLeu :TGAGGAGGTTTCG :TGAGGAGGTT ACATGGCGGTGCA TCGACCTGGC GCCGA GGTGCAGCCG A EGFR Lung 2137 9 2137 19 p.Gly719 :AAAAAGATCAAA :AAAAAGATCA →Ser GTGCTGGGCTCCG AAGTGCTGAG GTGCGTT CTCCGGTGCG TT FGFR3 Liver 1771 10 1771 20 p.Glu545 :ATCCTCTCTCTGA ATCCTCTCTC →Ala AATCACTGAGCAG TGAAATCACT GAGAAAG GCGCAGGAGA AAG

A more comprehensive but still partial list of mutations associated with cancer of various types is found at the website accessible from www.mycancergenom.org.

Alternately or in combination, in an aspect of the present disclosure, target sequences are determined de novo through tumor cell or infected sample sequencing. Samples are obtained and subjected to one or more of any number of targeted or untargeted sequencing approaches. Alleles or mutant loci are identified and selected for further analysis on the basis of their absence from a predicted healthy or host genome sequence. In some cases, the mutations or alleles are confirmed to be present exclusively in the tumor or in a subset of the tumor, such as a cell population implicated in tumor growth or metastasis.

The target sequence selection approaches are not mutually exclusive. For example, in some cases a treatment regimen comprises administration of a moiety having a guide RNA that targets a known tumor mutation. Upon observation that at least some symptoms are not ameliorated or that tumor morphology does not exhibit substantial change, or upon observation of any other sub-optimal outcome, one may proceed to sequence the remaining tumor tissue so as to identify at least one target sequence sufficient for a follow-on administration of a nuclease complex having a guide RNA or other sequence identification moiety that directs degradation of nucleic acids having the target sequence segment identified through the follow-on tumor sequencing.

The present disclosure provides a guide nucleic acid for use in a CRISPR/Cas or CRISPR PLUS™ system. A guide nucleic acid (e.g., guide RNA) can bind to a Cas protein and target the Cas protein to a specific location within a target polynucleotide. A guide nucleic acid can comprise a nucleic acid-targeting segment and a Cas protein binding segment.

A guide nucleic acid can be designed to target a pathway associated with or implicated in cancer. Non-limiting examples of pathways that can be targeted with a composition of the disclosure include apoptosis, β-catenin/Wnt signaling, cell cycle control, cellular architecture and microenvironment, chromatin remodeling/DNA methylation, cytotoxic chemotherapy mechanisms of action, DNA damage/repair, G-protein signaling, Hedgehog signaling, hormone signaling, immune checkpoints, JAK/STAT signaling, kinase fusions, MAP kinase signaling, metabolic signaling, PI3K/AKT1/MTOR, protein degradation/ubiquitination, receptor tyrosine kinase/growth factor signaling, RNA splicing, and TGFβ signaling.

A guide nucleic acid can be designed to target a pathway associated with or implicated in cancer. Non-limiting examples of genes that can be targeted with a composition of the disclosure include AKT1, ALK, BRAF, EGFR, HER2, KRAS, MEK1, MET, NRAS, PIK3CA, RET, and ROS1.

Specific targeting strategies of the present disclosure include targeting copy number variations (CNVs), which may be present in more abundance in cancer cells, targeting a structural variation (SV) junction such as CNVs, inversions, or translocations, wherein the junction sequence can span an SV locus and another normal locus, targeting indels (insertions/deletions) such as insertions, deletions, or single nucleotide polymorphisms (SNPs), or targeting integrated sequences such as viral DNA sequences integrated into a cell. Most cells have CNVs that can be targeted. Examples of cell lines with SV include the ERBB2 gene region in SK-BR-3 breast cancer cells. Examples of cell lines having indels include the EGFR gene in HCC827 and KRT16 gene in SK-BR-3. Examples of integrated sequences include cancer cells developed by integrative tumorigenic virus such as HPV (e.g., HPV sequence in HeLa cells) or HBV.

Identifying cancer sequences to target included downloading SVs, CNVs, Indels, and SNP data of cancer cell lines from CCLE and Harmonizome web databases. The expected copy number (N) of CNV can be calculated from copy number value (V) as follows; N=

2×2

{circumflex over ( )}V. Sequence data was downloaded and confirmed with human reference genome (GRCh38, hg19) from UCSC genome browser, NCBI Genbank and RefSeq database. If 5′-NGG-3′s, which is a PAM (protospacer adjacent motif) sequence of Streptococcus pyogenes Cas9 (SpCas9), are around cancer specific sequences, some of these 20 nucleotide sequences at 3′ direction of PAM were chosen and designated as CRISPR/Cas9 target spacer. Methods to select and design target sequences can meet the following three criteria: (1) G or C can be at both termini of sequences for high binding affinity of sgRNA, (2) GC contents (%) of sequences can be between 40˜60% for high binding affinity of sgRNA, and (3) A or T can be between 3 and 4 nucleotide of 3′ directed position of PAM, which is a cleavage site of SpCas9, for high cleavage efficiency of SpCas9.

A guide nucleic acid can be designed to target any of the following target sequences associated with specific genes in particular cell lines. TABLE 2 shows spacer sequence data table for Cas9 sgRNA. Copy number from CCLE database (portals.broadinstitute.org/ccle/data) & Harmonizome (amp.pharm.mssm.edu/Harmonizome). Indel from CCLE database. Target sequence & Locus from UCSC genome browser (genome.ucsc.edu/cgi-bin/hgGateway?redirect=manual&source=genome.ucsc.edu) & NCBI GenBank, RefSeq (www.ncbi.nlm.nih.gov/genbank, www.ncbi.nlm.nih.gov/refseq). *Indel mutation data were not secured in case of HeLa cancer cell line.

TABLE 2 Spacer sequence data table for Cas9 sgRNA. SEQ Locus Cell Gene Copy Essen- Target Target ID Exon| (hg19, Type line name # Indel tiality # Sequence NO: Intron NGG) Lung HCC827 EGFR >16 15 bp Oncogene 1 CGGAGATGTC 21 Exon chr7: DEL TTGATAGCGA 55,242,454 (−) VSTM2 >13 WT Non- 1 AGCTTCCTAGC 22 Intron chr7: A csscntial AAGTAACAG 54,610,610 (+) KIF5A >14 WT Non- 1 GCGCATCTTCC 23 Intron chr12: essential CTTTGTTAT 57,944,543 (+) H1563 IRX1 >8 WT Non- 1 GTCCGGAAGA 24 Intron chr5: essential GGAACTAGAA 3,596,613 (+) ADAMT >7 WT Non- 1 CTCCGTGCCGC 25 Intron chr5: S16 essential TGGTTTATT 5,140,952 (+) H1299 GNPDA >12 WT Oncogene 1 CAGAAGCTCT 26 Intron chr4: 2 GCATTCATCC 44,720,076 (−) KCNE2 >40 WT Non- 1 GGTGATGTGA 27 Intron chr21: essential GTTCTAGTCC 35,737,394 (+) SLC15A >12 WT Oncogene 1 GGACCGATTG 28 Intron chr12: 5 TGAGAAATGC 16,429,653 (−) SMIM11 >40 WT Non- 1 GTGCCCAGTGT 29 Intron chr21: essential GATGATATT 35,752,339 (+) A549 DACH2 >18 WT Non- 1 GCATGGCTTTT 30 Intron chrX: essential GGCTGTTCG 85,404,410 (+) HERC2P >8 WT Oncogene 1 GCTGTGATTTC 31 Intron chr15: 2 AACAGGACG 23,286,778 (−) CD68 >10 WT Oncogene 1 AGACCATTGG 32 Intron chr17: AGACTACACG 7,483,562 (+) SHBG >9 WT Non- 1 ATAGTACTAG 33 Intron chr17: essential GCTGCCTCAC 7,534,459 (+) Breast SKBR3 ERBB2 >33 WT Oncogene 8 CATGCTCCGCC 34 Exon chr17: ACCTCTACC 37,863,321 (+) CTGCAGCTTCG 35 Exon chr17: AAGCCTCAC 37,864,786 (+) CTTGTTGTGGT 36 Intron chr17: TTCTCAACC 37,863,519 (+) GGAAGACGCC 37 Intron chr17: CTCAGAAGAT 37,864,935 (+) GCCTGTAATCC 38 Intron chr17: CAGCTACTC 37,846,501 (+) CAGGCTAGAG 39 Intron chr17: TGAAATGGTG 37,880,586 (+) SCNA CTTCCTTGTAC 40 junc- N/A CAACACGTA tion CAGGTGTGTA 41 junc- N/A CCAACACGTA tion KRT16 >8 2 bp Non- 2 CCAGGAGTAC 42 Exon chr17: DEL, essential CAGCTGCCAT 39,766,652 1 bp (−) DEL 3 bp INS TCTTCACATCA 43 Exon chr17: TGCAGCAGC 39,766,661 (+) Colon HT29 LINC005 >9 WT Non- 1 AGTGGCCAGG 44 Intron chr8: 36 essential ATTGATTCAG 117,336,549 (+) TRPS1 >8 WT Non- 1 GACAGCAGAA 45 Intron chr8: essential TGACCTTGGT 116,630,763 (−) CDK8 >18 WT Oncogene 1 CCTGTCTTGTT 46 Intron chr13: CCCAGTCAT 26,960,973 (+) TRAPPC >13 WT Non- 1 GTAAGCTTACC 47 Intron chr8: 9 essential TAGAGACCC 141,460,868 (−) HERC2P >8 WT Oncogene 1 GCTGTGATTTC 31 Intron chr15: 2 AACAGGACG 23,286,778 (−) Pancreas Capan2 SIRPBI >24 WT Non- 1 GACACCCTAA 48 Intron chr20: essential CAGGGTTATG 1,600,151 (−) MRC1 >18 WT Non- 1 CTCACTGCAGC 49 Intron chr10: essential CTTGACATC 17,853,651 (+) ATP11A >8 WT Oncogene 1 CGTCCAAGTGT 50 Intron chr13: GTGAGTGAG 113,485,903 (+) POTEB >7 WT Non- 1 GTGGAAACCT 51 Intron chr15: essential CAGAGATGTC 22,082,219 (−) HERC2P >7 WT Oncogene 1 GCTGTGATTTC 31 Intron chr15: 2 AACAGGACG 23,286,778 (−) Cervix HeLa PRDM9 >8 Non- 1 GGACCCTATCT 52 Intron chr5: essential GAATGTGCA 23,509,294 (+) CDKN2 >10 Non- 1 GTGCATTCCAC 53 Intron chr15: B essential GCGTAAAAC 22,008,651 (−) HPV 30 Viral Viral 4 TGCTTATTGCC 54 viral N/A ACCACCTGC CCTGCAGGAA 55 viral N/A CCCTAAAATA CCATATTTTAG 56 viral N/A GGTTCCTGC TGCAGGTGGT 57 viral N/A GGCAATAAGC ETC LINE2 >100 WT Non- 1 AATCTCCCCCA 58 LINE N/A (mt2) essential CCCTTAAGA s CCR5 2 WT Non- 1 TGACATCAATT 59 Exon chr3: essential ATTATACAT 46,414,443 (+) HPRT1 2 WT House- 1 GCATTTCTCAG 60 Intron chrX: Keeping TCCTAAACA 133,607,78 (+) NT 0 N/A N/A 3 GGGTAACCGT 61 NT1 N/A GCGGTCGTAC GGGTAACCGT 62 NT2 N/A GGGTA NT3 N/A

Guide RNA can target sequences that appear multiple times in a particular gene, thereby resulting in multiple cuts upon administration of the guide RNA with Cas9. In other words, Cas9/guide RNA compositions disclosed herein induce cleavage of target sequences at multiple positions in a gene and induce cell death. For example, guide RNA can be used to target CCR5 (having two cut sites in certain cells), HPRT1 (having two cut sites in certain cells), MT2 (having over 100 cut sites in particular cells), SMIM11 (having over 40 cut sites in certain cells), GNPDA2 (having over 12 cut sites in particular cells), SLC15A5 (having over 12 cut sites in particular cells), and KCNE2 (having over 40 cut sites in particular cells). In some embodiments, controls include HPRT1 (a house keeping gene). CCR5, HPRT1, MT2, SMIM11, and KCNE2 can be non-essential genes. In particular embodiments, the guide RNA and Cas9 compostiions disclosed herein are oncogenic and target mutant sequences in cancer cells to induce efficient cell death.

Cells that can be targeted by the guide RNA and Cas9 compositions disclosed herein, to in some instances introduce multiple cuts resulting in cell death, include lung cancer cells, breast cancer cells, cervical cancer cells, colon cancer cells, ovarian cancer cells, and pancreatic cancer cells. Cell lines consistent with the compositions and methods disclosed herein include H1299 cells and H1563 cells. The guide RNA and Cas9 compostions disclosed herein are delivered to cells via a variety of methods. The compositions can be electroporated into cells, with or without Lipofectamine, and can also be delivered to cells using a ribonucleoprotein (RNP). Guide RNA and Cas9 compositions can also be delivered using expression vectors, such as the PX459 expression vector.

Illustrative guide RNA sequences are also provided in Table 6 and paragraph [00303] and Table 7 and paragraph [00305] for use with polypeptides comprising Cas12 and Cas9 nucleases, respectively.

In the present disclosure, a guide nucleic acid can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target polynucleotide in the genome of a cell. In one implementation, a guide nucleic acid can be RNA, for example, a guide RNA. In another implementation, a guide nucleic acid can be DNA. In still another implementation, a guide nucleic acid may include DNA and RNA. Further, a guide nucleic acid can be single stranded or double-stranded. Furthermore, a guide nucleic acid can comprise a nucleotide analog or a modified nucleotide. In an aspect of the present disclosure, the guide nucleic acid can be programmed or designed to bind to a sequence of nucleic acid site-specifically.

In another aspect of the present disclosure, a guide nucleic acid can comprise one or more modifications to provide the nucleic acid with a new or enhanced feature. By way of example, a guide nucleic acid can comprise a nucleic acid affinity tag. Further, a guide nucleic acid can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.

In an aspect of the present disclosure, the guide nucleic acid can comprise a nucleic acid-targeting region (e.g., a spacer region), for example, at or near the 5′ end or 3′ end, that is complementary to a protospacer sequence in a target polynucleotide. The spacer of a guide nucleic acid can interact with a protospacer in a sequence-specific manner via hybridization (base pairing). The protospacer sequence can be located 5′ or 3′ of protospacer adjacent motif (PAM) in the target polynucleotide. The nucleotide sequence of a spacer region may vary and determine the location for cleavage within the target nucleic acid with which the guide nucleic acid can interact. The spacer region of the guide nucleic acid may be designed or modified to hybridize to any desired sequence within the target nucleic acid.

In another aspect of the present disclosure, a guide nucleic acid can comprise two separate nucleic acid molecules, which can be referred to as a double guide nucleic acid or a single nucleic acid molecule, which can be referred to as a single guide nucleic acid (e.g., sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA but lacking a tracRNA. In some embodiments, the guide nucleic acid is a double guide nucleic acid comprising non-fused crRNA and tracrRNA. An exemplary double guide nucleic acid can comprise a crRNA-like molecule and a tracrRNA-like molecule. An exemplary single guide nucleic acid can comprise a crRNA-like molecule. An exemplary single guide nucleic acid can comprise a fused crRNA-like molecule and a tracrRNA-like molecule.

A crRNA can comprise the nucleic acid-targeting segment (e.g., spacer region) of the guide nucleic acid and a stretch of nucleotides that can form one half of a double-stranded duplex of the Cas protein-binding segment of the guide nucleic acid.

A tracrRNA can comprise a stretch of nucleotides that forms the other half of the double-stranded duplex of the Cas protein-binding segment of the gRNA. A stretch of nucleotides of a crRNA can be complementary to and hybridize with a stretch of nucleotides of a tracrRNA to form the double-stranded duplex of the Cas protein-binding domain of the guide nucleic acid.

The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The crRNA can also provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., protospacer). The sequence of a crRNA, including spacer region, or tracrRNA molecule can be designed to be specific to the species in which the guide nucleic acid is to be used.

In some embodiments, the nucleic acid-targeting region of a guide nucleic acid can be between 18 to 72 nucleotides in length. The nucleic acid-targeting region of a guide nucleic acid (e.g., spacer region) can have a length of from about 12 nucleotides to about 100 nucleotides. For example, the nucleic acid-targeting region of a guide nucleic acid (e.g., spacer region) can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 12 nt to about 18 nt, from about 12 nt to about 17 nt, from about 12 nt to about 16 nt, or from about 12 nt to about 15 nt. Alternatively, the DNA-targeting segment can have a length of from about 18 nt to about 20 nt, from about 18 nt to about 25 nt, from about 18 nt to about 30 nt, from about 18 nt to about 35 nt, from about 18 nt to about 40 nt, from about 18 nt to about 45 nt, from about 18 nt to about 50 nt, from about 18 nt to about 60 nt, from about 18 nt to about 70 nt, from about 18 nt to about 80 nt, from about 18 nt to about 90 nt, from about 18 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about 20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20 nt to about 100 nt. The length of the nucleic acid-targeting region can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The length of the nucleic acid-targeting region (e.g., spacer sequence) can be at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

In some embodiments, the nucleic acid-targeting region of a guide nucleic acid (e.g., spacer) is 20 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 19 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 18 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 17 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 16 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 21 nucleotides in length. In some embodiments, the nucleic acid-targeting region of a guide nucleic acid is 22 nucleotides in length.

The nucleotide sequence of the guide nucleic acid that is complementary to a nucleotide sequence (target sequence) of the target nucleic acid can have a length of, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt. The nucleotide sequence of the guide nucleic acid that is complementary to a nucleotide sequence (target sequence) of the target nucleic acid can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt.

A protospacer sequence can be identified by identifying a PAM within a region of interest and selecting a region of a desired size upstream or downstream of the PAM as the protospacer. A corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

A spacer sequence can be identified using a computer program (e.g., machine readable code). The computer program can use variables such as predicted melting temperature, secondary structure formation, and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence, methylation status, presence of SNPs, and the like.

The percent complementarity between the nucleic acid-targeting sequence (e.g., spacer sequence) and the target nucleic acid (e.g., protospacer) can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%. The percent complementarity between the nucleic acid-targeting sequence and the target nucleic acid can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% over about 20 contiguous nucleotides.

The Cas protein-binding segment of a guide nucleic acid can comprise two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another. The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another can be covalently linked by intervening nucleotides (e.g., a linker in the case of a single guide nucleic acid). The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another can hybridize to form a double stranded RNA duplex or hairpin of the Cas protein-binding segment, thus resulting in a stem-loop structure. The crRNA and the tracrRNA can be covalently linked via the 3′ end of the crRNA and the 5′ end of the tracrRNA. Alternatively, tracrRNA and crRNA can be covalently linked via the 5′ end of the tracrRNA and the 3′ end of the crRNA.

The Cas protein binding segment of a guide nucleic acid can have a length of from about 10 nucleotides to about 100 nucleotides, e.g., from about 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt. For example, the Cas protein-binding segment of a guide nucleic acid can have a length of from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.

The dsRNA duplex of the Cas protein-binding segment of the guide nucleic acid can have a length from about 6 base pairs (bp) to about 50 bp. For example, the dsRNA duplex of the protein-binding segment can have a length from about 6 bp to about 40 bp, from about 6 bp to about 30 bp, from about 6 bp to about 25 bp, from about 6 bp to about 20 bp, from about 6 bp to about 15 bp, from about 8 bp to about 40 bp, from about 8 bp to about 30 bp, from about 8 bp to about 25 bp, from about 8 bp to about 20 bp or from about 8 bp to about 15 bp. For example, the dsRNA duplex of the Cas protein-binding segment can have a length from about from about 8 bp to about 10 bp, from about 10 bp to about 15 bp, from about 15 bp to about 18 bp, from about 18 bp to about 20 bp, from about 20 bp to about 25 bp, from about 25 bp to about 30 bp, from about 30 bp to about 35 bp, from about 35 bp to about 40 bp, or from about 40 bp to about 50 bp.

In some embodiments, the dsRNA duplex of the Cas protein-binding segment may have a length of 36 base pairs. The percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment can be at least about 60%. For example, the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment can be at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In some cases, the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment is 100%.

The linker (e.g., that links a crRNA and a tracrRNA in a single guide nucleic acid) can have a length of from about 3 nucleotides to about 100 nucleotides. For example, the linker can have a length of from about 3 nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt) to about 80 nt, from about 3 nucleotides (nt) to about 70 nt, from about 3 nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) to about 50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3 nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about 20 nt or from about 3 nucleotides (nt) to about 10 nt. For example, the linker can have a length of from about 3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt. In some embodiments, the linker of a DNA-targeting RNA is 4 nt.

As noted above, in an aspect of the present disclosure, guide nucleic acids can include modifications or sequences that provide for additional desirable features (e.g., modified or regulated stability; subcellular targeting; tracking with a fluorescent label; a binding site for a protein or protein complex; and the like). Examples of such modifications include, for example, a 5′ cap (a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail (a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and/or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (a hairpin)); a modification or sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, and so forth); a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyl transferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and combinations thereof.

In another aspect of the present disclosure, a guide nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A guide nucleic acid can comprise a nucleic acid affinity tag. A nucleoside can be a base-sugar combination. The base portion of the nucleotide can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. In forming guide nucleic acids, the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Further, within guide nucleic acids, the phosphate groups may commonly be referred to as forming the internucleoside backbone of the guide nucleic acid. The linkage or backbone of the guide nucleic acid can be a 3′ to 5′ phosphodiester linkage.

In another aspect of the present disclosure, a guide nucleic acid can comprise a modified backbone and/or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

Suitable modified guide nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, a 5′ to 5′ or a 2′ to 2′ linkage. Suitable guide nucleic acids having inverted polarity can comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage (such as a single inverted nucleoside residue in which the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms can also be included.

A guide nucleic acid can comprise one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2- (a methylene (methylimino) or MMI backbone), —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and —O—N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2-).

A guide nucleic acid can comprise a morpholino backbone structure. For example, a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.

A guide nucleic acid can comprise polynucleotide backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

A guide nucleic acid can comprise a nucleic acid mimetic. The term “mimetic” can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. The backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

A guide nucleic acid can comprise linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomeric units in a morpholino nucleic acid. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholino-based polynucleotides can be non-ionic mimics of guide nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. The incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. A further modification can include Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (—CH2-), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation and good solubility properties.

A guide nucleic acid can comprise one or more substituted sugar moieties. Suitable polynucleotides can comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO) mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. A sugar substituent group can be selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an guide nucleic acid, or a group for improving the pharmacodynamic properties of an guide nucleic acid, and other substituents having similar properties. A suitable modification can include 2′-methoxyethoxy (2′-O—CH2 CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE, an alkoxyalkoxy group). A further suitable modification can include 2′-dimethylaminooxyethoxy, (a O(CH2)2ON(CH3)₂ group, also known as 2′-DMAOE), and 2′-dimethylaminoethoxyethoxy (also known as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), 2′-O-CH2-O-CH2-N(CH3)2.

Other suitable sugar substituent groups can include methoxy (—O—CH3), aminopropoxy CH2 CH2 CH2NH2), allyl (—CH2-CH═CH2), —O-allyl CH2-CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked nucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

A guide nucleic acid may also include nucleobase (often referred to simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases can include the purine bases, (e.g. adenine (A) and guanine (G)), and the pyrimidine bases, (e.g. thymine (T), cytosine (C) and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino¬adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H¬pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties can include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases can be useful for increasing the binding affinity of a polynucleotide compound. These can include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions can increase nucleic acid duplex stability by 0.6-1.2° C. and can be suitable base substitutions (e.g., when combined with 2′-O-methoxyethyl sugar modifications).

A modification of a guide nucleic acid can comprise chemically linking to the guide nucleic acid one or more moieties or conjugates that can enhance the activity, cellular distribution or cellular uptake of the guide nucleic acid. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups can include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that can enhance the pharmacokinetic properties of oligomers. Conjugate groups can include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that can enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a nucleic acid. Conjugate moieties can include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

A modification may include a Protein Transduction Domain or PTD (a cell penetrating peptide (CPP)). The PTD can refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD can be attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, and can facilitate the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. A PTD can be covalently linked to the amino terminus of a polypeptide. A PTD can be covalently linked to the carboxyl terminus of a polypeptide. A PTD can be covalently linked to a nucleic acid.

Exemplary PTDs can include, but are not limited to, a minimal peptide protein transduction domain; a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines (SEQ ID NO: 234)), a VP22 domain, a Drosophila Antennapedia protein transduction domain, a truncated human calcitonin peptide, polylysine, and transportan, arginine homopolymer of from 3 arginine residues to 50 arginine residues (SEQ ID NO: 234). The PTD can be an activatable CPP (ACPP). ACPPs can comprise a polycationic CPP (e.g., Arg9 or “R9” (SEQ ID NO: 63)) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9” (SEQ ID NO: 64)), which can reduce the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion can be released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.

An exemplary chimeric polypeptide of the present disclosure comprises a nuclease such as a site-specific endonuclease or a domain thereof. Non-limiting exemplary site-specific endonucleases that are suitable with the present disclosure may include but are not limited to CRISPR-associated (Cas) polypeptides or Cas nucleases including Class 1 Cas polypeptides, Class 2 Cas polypeptides, type I Cas polypeptides, type II Cas polypeptides, type III Cas polypeptides, type IV Cas polypeptides, type V Cas polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivative thereof; any variant thereof; and any fragment thereof.

In some cases, a chimeric polypeptide of the present disclosure comprises one or more domains of a site-specific endonuclease. Non-limiting examples of domains suitable for use with the present disclosure may include guide nucleic acid recognition or binding domain; nuclease domains such as DNase domain, RNase domain, RuvC domain, and HNH nuclease domain; DNA binding domain; RNA binding domain; helicase domains; protein-protein interaction domains; and dimerization domains. A guide nucleic acid recognition or binding domain interacts with a guide nucleic acid. A nuclease domain comprises catalytic activity for nucleic acid cleavage. Alternatively, a nuclease domain may be a mutated nuclease domain that lacks or has reduced catalytic activity. A site-specific endonuclease may be a chimera of various site-specific endonuclease proteins, for example, comprising domains from different Cas proteins.

In some cases, a site-specific endonuclease of the present disclosure is a wild-type form of the protein. Alternatively, a site-specific endonuclease is a modified version of the wildtype form, for example, comprising an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof, relative to a wild-type version of the protein.

In some cases, a site-specific endonuclease of the disclosure comprises a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a wild type exemplary site-specific endonuclease.

In some cases, a site-specific endonuclease of the disclosure comprises an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain, for example, a RuvC domain or an HNH domain, of a wild-type site-specific endonuclease.

Proteins that bind to and are guided by a guide RNA to direct sequence specific cleavage or that, otherwise, bind nucleic acid sequences in a sequence specific way to trigger non-specific cleavage are consistent with the present disclosure. Said proteins include programmable endonucleases, such as programmable Cas endonucleases. In some cases, a site-specific endonuclease of the disclosure comprises a Cas polypeptide or a domain thereof. Non-limiting exemplary Cas polypeptides suitable for use with the present disclosure include Cas9, Cpf1 (or Cas12a), c2c1, C2c2 (or Cas13a), Cas13, Cas13a, Cas13b, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966; any derivative thereof; any variant thereof; and any fragment thereof.

In some cases, a site-specific endonuclease of the present disclosure comprises inducible non-specific nuclease activity or promiscuous activity in addition to target-specific activity. The non-specific activity may be activated by site-specific cleavage of the target nucleic acid by the site-specific endonuclease. Non-limiting exemplary site-specific endonucleases comprising inducible non-specific nuclease activity may include Cas12a (Cpf1), Cas13, C13a, and Cas13b.

In some cases, a site-specific endonuclease may include a Cas9 polypeptide, including any derivative thereof; any variant thereof; and any fragment thereof. Cas9 is classified as a class II, Type II CRISPR/Cas effector protein. An exemplary Cas9 polypeptide is Cas9 from Streptococcus pyogenes, referred to herein as SpCas9, which is composed of 1,368 amino acids. Cas9 is characterized to have two endonuclease domains, HNH and RuvC, and a recognition lobe (REC) domain. The HNH domain cleaves the DNA strand complementary to the guide RNA sequence. The RuvC-like domain cuts the other non-complementary DNA strand through Watson-Crick base pairing formed by a guide RNA/Cas9 complex.

In some cases, a Cas9 protein comprises mutations. For example, substitution of aspartic acid (D) at the 10^(th) amino acid in the RuvC domain to alanine (A) removes the RuvC-dependent nuclease function leaving only HNH-dependent endonuclease function. The D10A variant of Cas9, known as a nickase, can be used to generate a single strand nick at the target site. An additional substitution mutation, a change from histidine (H) to alanine (A) at the 840^(th) amino acid in HNH domain of Cas9 H840A, produces a deactivated Cas9 protein lacking all nuclease activity. The deactivated Cas9, comprising mutations D10A and H840A, retains sequence-specific binding function and can serve as a functional transcription factor, for example, when fused in frame with either an activator or a repressor domain.

Proteins that bind to and are guided by a guide RNA to direct sequence specific cleavage or that, otherwise, bind nucleic acid sequences in a sequence specific way to trigger non-specific cleavage are consistent with the present disclosure. Said proteins include programmable endonucleases, such as programmable Cas endonucleases. Examples of programmable Cas endonucleases consistent with the present disclosure include Cas12a (or Cpf1), Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas14, Cas9, or others. In some cases, a site-specific endonuclease may include Cas12a (Cpf1), including any derivative thereof; any variant thereof; and any fragment thereof. Cas12a is classified as a class II, Type V CRISPR/Cas effector protein having about 1,300 amino acids. Cas12a is smaller than Cas9. Cas12a comprises two major domains such as REC and RuvC domains. Cas12a lacks the HNH endonuclease domain as in Cas9. Cas12a cleaves a double stranded DNA (dsDNA) immediately downstream from T-rich (5′-TTTN-3′) PAM. Cas12a generates a 4-5 nt-long 5′-overhang 20 nucleotides away from T-rich PAM. In some cases, the sticky ends produced by Cas12a enhance the efficiency of DNA replacement during HR.

In some cases, a site-specific endonuclease may include Cas13a (C2c2). Alternatively, in some cases, a site-specific endonuclease may include Cas13b. Cas13 is an RNA-targeting endonuclease that exhibits a collateral effect of promiscuous RNAs activity upon target recognition.

In some cases, a site-specific endonuclease such as a Cas polypeptide may be from the organism Streptococcus pyogenes (S. pyogenes). Alternatively, a number of eubacterial or other microbial organisms may be suitable sources for site specific endonucleases such as Cas polypeptides. Source organisms are selected by any number of criteria, such as GC bias or codon bias of their encoded proteins, optimal growth temperature (which often corresponds to enzyme optimal activity), availability, presence of regulatory sites, or other relevant criteria. Any of the following non-limiting examples are suitable for use as a source of the site-specific endonuclease: Leptotrichia wadei, Leptotrichia shahii, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Prevotella, or Francisella novicida. Other source organisms are consistent with the present disclosure herein.

Some exemplary chimeric polypeptides of the present disclosure may comprise an exonuclease or a domain thereof. Non-limiting examples of exonucleases suitable for use with the present disclosure include exonucleases such as 5′-3′ exonucleases, 3′-5′ exonucleases, and 5′-3′ alkaline exonucleases. Table 3 shows exemplary exonucleases suitable for fusion with a site-specific endonuclease to generate a chimeric polypeptide of the present disclosure.

TABLE 3 Exemplary Exonucleases Added Origin of added moieties domain Function of added moieties RecE Escherichia coli 5′→3′ exonuclease RecJ Escherichia coli 5′→3′ exonuclease T5 Bacteriophage T5 5′→3′ exonuclease Exo I Prokaryote & 3′→5′ exonuclease enukaryote Exo III Escherichia coli 3′→5′ exonuclease Exo VII Escherichia coli Both 5′→3′ and 3′→5′ directions. Lexo Escherichia coli Lambda exonuclease, 5′→3′ exonuclease RecBCD Escherichia coli Both 5′→3′ and 3′→5′ directions Exo T Escherichia coli 3′→5′ exonuclease Trex1 Homo sapiens 3′→5′ exonuclease Trex2 Homo sapiens 3′→5′ exonuclease Mungbean Vigna radiata Single strand DNA digestion

In an exemplary chimeric polypeptide, the exonuclease comprises a 5′-3′ exonuclease or a catalytic domain thereof. Non-limiting examples of 5′-3′ exonucleases suitable for use with the disclosure include those from prokaryotes such as RecE, RecJ, RexB, and the exonuclease domain of DNA Polymerase I; bacteriophages such as T2, T3, T4, T5, T7, or lambda bacteriophage; and eukaryotes such as Xrn1 or Exo1 5′-exonuclease. In some cases, the mutation efficiency achieved with a chimeric polypeptide comprising 5′-3′ exonuclease activity is greater than that achieved with a chimeric polypeptide comprising 3′-5′ exonuclease activity.

In an exemplary chimeric polypeptide, the exonuclease may comprise RecJ or a catalytic domain thereof. RecJ is a processive monomeric exonuclease of about 60 kD. RecJ degrades ssDNA in a 5′ to 3′ polarity in a reaction that requires Mg2+, resulting in degradation of DNA to mononucleotides. RecJ nuclease can produce ssDNA with 3′ overhang tails which may be bound by a single-stranded DNA binding protein. In some cases, RecJ does not require a terminal 5′ phosphate and will digest equally well DNA terminating in 5′ OH. In some cases, RecJ has no activity on blunt dsDNA. In some cases, RecJ requires at least 6 unpaired bases to bind and to initiate degradation. Once bound to a ssDNA-tailed molecule, RecJ can digest into a dsDNA region to a limited extent but most often terminates digestion at the ds/ssDNA boundary. RecJ is specialized for degradation from a single-strand gap, then leads to produce 3′ ssDNA tailed recombinogenic molecules from double-strand ends. RecJ can remove 5′ overhanging ssDNA produced by 3′ to 5′ exonuclease activity to increase the indel mutation rate. In some cases, RecJ is suited to link to C-terminus of a site-specific endonuclease such as Cpf1, for example, because RecJ is capable of chewing 5-nt overhang produced by Cpf1.

In an exemplary chimeric polypeptide, the exonuclease may comprise RecE or a domain thereof. RecE (also known as Exonuclease VIII or ExoVIII) possesses processive Mg2+-dependent 5′ to 3′ exonuclease activity. RecE is the functional equivalent to the lambda exonuclease. An exemplary source of RecE is E. coli. RecE is composed of about 866 amino acids. RecE resects double stranded DNA from 5′ to 3′ direction. RecE requires a 5′ phosphate for its exonuclease activity. In some cases, RecE preferentially acts on dsDNA blunt ends, which are produced by site-specific endonucleases such as Cas9.

In an exemplary chimeric polypeptide, the exonuclease may comprise T5 exonuclease or a catalytic domain thereof. T5 exonuclease (T5-exo) is a 276 amino acid protein. T5 exonuclease has a 5′→3′ exodeoxyribonuclease activity. T5 exonuclease degrades ssDNA or dsDNA in the 5′ to 3′ direction. T5 exonuclease is able to initiate nucleotide removal from the 5′ termini or at gaps and nicks of linear or circular dsDNA. Some T5 exonuclease moieties do not degrade supercoiled circular dsDNA. T5 exonuclease can enhance indel mutation and HR by producing 3′-overhangs from double strand breaks generated by a site-specific endonuclease such as Cas9.

In an exemplary chimeric polypeptide, the exonuclease may comprise Lambda exonuclease or a catalytic domain thereof. Lambda exonuclease belongs to exonuclease IV, which has exonucleolytic cleavage in the 5′- to 3′-direction to yield nucleoside 5′-phosphates. Lambda exonuclease has a preference for blunt-ended, 5′-phosphorylated dsDNA. Lambda Exonuclease is unable to initiate DNA digestion at nicks or gaps. Lambda Exonuclease can enhance both the indel mutation rate and HR by producing 3′ overhangs from Cas9-produced blunt ends.

In an exemplary chimeric polypeptide, the exonuclease may comprise an exonuclease domain of DNA polymerase I. Cleavage of DNA polymerase I, for example, by the protease subtilisin, produces a small fragment and a large fragment (also known as the Klenow fragment). The small fragment, referred to herein as small fragment of DNA pol I, comprises 5′→3′ exonuclease and 5′ flap (5′ overhang extending from duplex strands) endonuclease activity, and is suitable for use as a DME comprising 5′→3′ exonuclease activity. The larger fragment comprises 5′→3′ polymerase activity, and 3′→5′ exonuclease activity.

In an exemplary chimeric polypeptide, the exonuclease may comprise a 3′-5′ exonuclease or a catalytic domain thereof. Non-limiting examples of 3′-5′ exonucleases suitable for use with the present disclosure may include TREX such as TREX2, Mungbean nuclease, Exonuclease I, Exonuclease III, Exonuclease VII, and RecBCD exonuclease.

In an exemplary chimeric polypeptide, the exonuclease may comprise TREX or a catalytic domain thereof. The TREX enzyme can be TREX1 or TREX2. TREX2 is an autonomous eukaryotic exonuclease, which is a 279 amino acid protein. The TREX1 and TREX2 proteins form homodimers with 3′ excision activities. TREX1 and TREX2 employ single-stranded oligonucleotides, and most closely relate structurally with the bacterial epsilon subunit of DNA pol III, Exo1, and ExoX.

In an exemplary chimeric polypeptide, the exonuclease may comprise Mungbean nuclease or a catalytic domain thereof. Mungbean nuclease is a 355 amino acid protein, isolated from the sprouts of the mung bean, Vigna radiata. Mungbean nuclease cleaves nucleotides in a step-wise manner from ssDNA or a flap structure of dsDNA with a free single-stranded 5′-end. In some cases, Mungbean exonuclease does not digest double-stranded DNA, double-stranded RNA, DNA/RNA hybrids, or the intact strand of nicked duplex DNA. Mungbean nuclease catalyzes the specific degradation of single-stranded DNA or RNA, and produces mono and oligonucleotides carrying a 5′-P terminus. In some cases, Mungbean exonuclease increases indel mutation by removing 5′ and 3′ single-stranded overhangs produced by site-specific endonucleases such as Cas9.

In an exemplary chimeric polypeptide, the exonuclease may comprise Exonuclease I (Exo I) or a catalytic domain thereof. Exonuclease I is an 846 amino acid protein with a Mg²⁺-dependent 3′ to 5′ single strand exonuclease activity. Exonuclease I digests dsDNA to mononucleotides. Exonuclease I is a member of the DnaQ superfamily and its structure is similar to the 3′ to 5′ exonucleases active site of the Klenow fragment of DNA polymerase I. In some cases, Exonuclease I increases the indel mutation by removing single stranded overhang in double strand break ends produced by site-specific endonucleases such as Cas9.

In an exemplary chimeric polypeptide, the exonuclease may comprise exonuclease III (ExoIII) or a catalytic domain thereof. Exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of double-stranded DNA. Exemplary substrates of exonuclease III are blunt or recessed 3′-termini. In some cases, Exonuclease III also acts at nicks in duplex DNA to produce single-strand gaps, which can be resistant to cleavage, because often Exonuclease III may not be active on 3′-overhang termini with extensions 4 bases or longer being frequently resistant to cleavage. Exonuclease III produces unidirectional deletions from a linear molecule with one 3′-overhang resistant and one 5′-overhang or blunt susceptible terminus. Exonuclease III activity depends partially on the DNA helical structure and displays sequence dependence (C>A=T>G). Exonuclease III can also comprise RNase H, 3′-phosphatase and AP-endonuclease activities. In some cases, Exonuclease III increases indel mutations by accelerating the 3′ to 5′ exonuclease activity of Cas9.

In an exemplary chimeric polypeptide, the exonuclease may comprise exonuclease VII or a catalytic domain thereof. Exonuclease VII (ExoVII) cleaves single-stranded DNA from either 5′-3′ or 3′-5′ direction to yield 5′-phosphomononucleotides. Exonuclease VII also comprises an N-terminal DNA binding domain. In some cases, Exonuclease VII increases indel mutations by removing single stranded overhangs generated by Cas9-producing break ends.

In an exemplary chimeric polypeptide, the exonuclease may comprise RecBCD exonuclease or a catalytic domain thereof. RecBCD exonuclease comprises a Mg²⁺-dependent RecB domain, RecC domain, and RecD domain, which unwind dsDNA and degrade single-stranded DNA and double-stranded DNA. RecBCD cleaves ssDNA from either 5′-3′ or 3′-5′ direction to yield 5′-phosphomononucleotides. In some cases, RecBCD increases indel mutation by removing single-stranded overhangs generated in Cas9-based double strand breaks.

In another example, the exonuclease may comprise Exonuclease T or a catalytic domain thereof. Exonuclease T (Exo T) includes a 215 amino acid protein, a member of the DnaQ-like 3′-5′ exonucleases, with a DEDDh domain that contains four acidic DEDD residues (D23, E25, D125, and D186) for binding of two magnesium ions, and a histidine residue (H181) for functioning in the active site of 3′-hydroxyl terminus. Exonuclease T trims the 3′ end of structured DNA, including bulge, bubble, and Y-structured DNA.

Routes of administration. Nuclease ribonucleoprotein complexes may be administered through a number of administration routes consistent with the present disclosure herein. Administration is in some cases systemic, through oral administration of a formulation configured to be resistant to digestive uptake. Alternately, systemic administration is achieved through inhalation or through intravenous administration of a complex, such as through introduction of a formulation into the blood stream via injection or via intravenous drip.

Local administration may also be consistent with the present disclosure herein. Compositions are injected, topically administered at a site of affected cells as part of a salve or as a constituent of a patch, for example.

Administration is often impacted by the type of nucleic acids to be targeted. Melanomas, squamous cell carcinomas, skin infections or other accessible surface disorders are optionally subjected to topical or systemic administration. Palpable tumors may be subjected to topical administration or to injection of pre-assembled complexes at or near a tumor site, or may be subjected to systemic administration. Similarly, disorders involving lungs may be addressed through inhalation or through systemic delivery. Internal disorders, disorders involving remote tissues, or conditions where metastasis is suspected such that a cancer cell population is not expected to be localized at a single site are likely to be addressed using a delivery route that comprises at least some systemic delivery.

Further, in an aspect of the present disclosure, the administration may be performed via using nanoparticles as delivery platforms. By way of example, the administration may be done by using for example PEGylated Gold Nanoparticles, anionic gold nanoparticles, and certain polymers for systematic administration non-immune applications, and the administration may be done by using for example lipid-based nanoparticles and certain polymers for immune, vaccines and other applications wherein immune-stimulation is desired. In each of the delivery platforms, both immunotoxicity of both active pharmaceutical ingredient (API) and nanocarrier may be considered and an appropriate delivery system may be selected for a particular application. That is, when immunomodulation is desired, immunologically reactive carrier is used, and when immunoactivity is not desired, the immunologically reactive carrier is not used. As such, nanotechnology-formulated drugs may be developed and used for the present disclosure, based on pre-clinical immunotoxicity studies, for example, available in Dobrovolskaia M, J Control Release. 2015 Dec. 28; 220(0 0): 571-583 (ncbi.nlm.nih.gov/pmc/articles/PMC4688153/), which is incorporated herein in its entirety.

Multiple routes of administration to address a single disorder, performed concurrently or in sequence, are also consistent with the disclosure herein.

Temporal regulation of nuclease activity. As mentioned above, nuclease sequence specificity is optionally accompanied by temporal regulation of nuclease activity so as to further reduce off-target or other side-effects of administration. In an aspect of the present disclosure, temporal regulation is achieved through temporal, specific inactivation of nuclease complexes by at least one of enzymatic inactivation and sequestration via antibody binding. Temporal inactivation conveys the benefit of clearing the nuclease from an individual, either locally or systemically, so as to reduce the risk of off-target activity, particularly in situations where targeted nucleic acids are largely or completely cleared from an individual or a particular site at an individual.

As discussed above, some compositions and methods comprise temporal regulation of activity through administration of a treatment or composition that impacts at least one of nucleic acid recognition, endonucleolytic activity and exonucleolytic activity or other activity consistent with the present disclosure herein. An individual is subjected to a method or administered a composition consistent with the present disclosure herein and is administered a concurrent or follow-on composition or treatment so as to inactivate the nuclease-activity composition. An exemplary inactivating composition includes, but not limited to, ZnSO₄ or other ionic or nonionic molecule that impacts sequence identification or nuclease activity. Administration of such a composition impacts nuclease activity such that, even if it remains intact locally or systemically, the nuclease activity is reduced.

By way of example, a “quencher solution” or “stopper solution” as used in the present disclosure may include a composition that inhibits the nuclease activity and the quencher solution may include such as ZnSO4 or other ionic or nonionic modules. For example, depending upon a specific application, a quencher solution may be selected from various quencher solutions available as shown in Table 4, attached hereto, providing a possible combination table for prospective quencher solutions. In Table 4, columns stand for twelve anions (e.g., sulfate, nitrate, chloride, ammonium, citrate, arsenate, phosphate, ammonium phosphate, carbonate, fluoride, oxalate, hydroxide), which are able to ion-bind to metal cations, and rows stand for seven metal ions (e.g., Aluminum, Zinc, Iron, Lead, Copper, Silver, and Gold). Each combination between anions and metal ions is a prospective quencher to terminate CRISPR or CRISPR PLUS™ performance. All 84 quencher solutions are generated from seven metals and twelve anions. Less reactive metal cations, aluminum, zinc, iron, lead, copper, silver, and gold can displace magnesium, which is a crucial cofactor of CRISPR or CRISPR PLUS™ endonuclease activity to perform DNA cleavage reaction. Anions are available to deplete magnesium from Cas9 protein by binding to magnesium.

Table 4, below, illustrates that the metals (e.g., aluminum, zinc, iron, lead, copper, silver, and gold) can be combined with any of the following anions: sulfate (SO₄), nitrate (NO3), chloride (Cl), ammonium (NH₄), citrate (C₆H₈O₇), arsenate (AsO₄), phosphate (PO₄), ammonium phosphate (NH₄)₃PO₄, carbonate (CO₃), fluoride (F), oxalate (C₂O₄), and hydroxide (OH).

TABLE 4 A combination table for prospective quencher solutions Anions Sulfate Nitrate Chloride Ammonium Citrate Arsenate Phosphate Metals (SO₄) (NO₃) (Cl) (NH₄) (C₆H₈O₇) (AsO₄) (PO₄) Aluminum available available available available available available available Zinc available available available available available available available Iron available available available available available available available Lead available available available available available available available copper available available available available available available available Silver available available available available available available available Gold available available available available available available available Anions ammonium phosphate carbonate fluoride Oxalate Hydroxide Metals (NH₄)₃PO₄ (CO₃) (F) (C₂O₄) (OH) Aluminum available available available available available Zinc available available available available available Iron available available available available available Lead available available available available available copper available available available available available Silver available available available available available Gold available available available available available

Additional embodiments include but are not limited to endonuclease-killing nucleic acids that stably bind the nuclease such as CRISPR/Cas or CRISPR PLUS™ complex, rendering it unable to processively degrade nucleic acid substrate in a target-directed manner.

In an aspect of the present disclosure, both inactivation and sequestration of nuclease nucleoprotein complexes may be accomplished through administration of at least one complex-binding antibody. Such an antibody may, in various embodiments, block or impair substrate binding, block or interfere with endonuclease activity, block or interfere with exonuclease activity, or sequester ribonuclear complexes such that they are removed from the vicinity of nucleic acids in a tissue. Accordingly, an antibody may bind and impact, up to and including inactivating, a complex responsible for specific nucleic acid identification or targeted degradation.

In some cases, such a composition may be administered locally at a site of a tumor being targeted for selective nucleic acid degradation, either through injection, topical administration, or co-administration with the nuclease, optionally though a time-delayed release mechanism such as those known in the art. Alternately or in combination, such a composition is administered systemically, optionally in a sequestered from so as to be released or rendered available for activity in a limited range of tissues.

Alternately or in combination, local inactivation of nuclease is achieved through local heat administration at a tumor or other nuclease activity site. Heat administration in various cases may decrease nucleic acid base pairing efficiency, reduce nuclease ribonucleoprotein complex stability, or reduce nuclease ribonucleoprotein activity.

Co-administration and ongoing monitoring or treatment regimens. Nuclease compositions disclosed herein, such as ribonucleoprotein complexes, are administered to an individual as part of a stand-alone treatment regimen or in combination with at least one additional treatment regimen. In particular, an individual suspected or diagnosed as having a particular cancer is often administered a standard of care chemotherapeutic or radiotherapeutic as a first line of treatment, and is administered a composition as disclosed herein as a follow-on or secondary treatment. Such a treatment is in some cases to address deficiencies in an outcome from a primary treatment, or to serve as a confirmatory treatment to increase the likelihood of tumor or disease cell clearance, reducing the chance of relapse after remission.

Also contemplated is administration of a specific nuclease as disclosed herein concurrently with a frontline or conventional treatment, in some cases under conditions so as to convey a benefit of the conventional treatment but at a dose that reduces the side effects of the frontline treatment.

Composition administration in concert with or subsequent to a non-pharmaceutical treatment regimen, such as a surgical intervention is also contemplated, as is administration in combination with an antibiotic regimen, an exercise regimen or a modification to diet.

Alternately, in some cases a nuclease composition herein is administered as a frontline or initial treatment, so as to ameliorate or eliminate the side effects that often accompany chemotherapeutic or radiotherapeutic treatment regimens currently in use as cancer therapeutics.

Also contemplated herein is composition administration in combination with inhibitors of DNA double strand break repair. Inhibitors contemplated for use include small molecule inhibitors and macromolecular inhibitors, for example, a ribonucleoprotein complex comprising a guide nucleic acid that targets a gene associated with DNA double strand break repair such as BRCA gene or BRCA pathway. Inhibitors can be administered concurrently with, prior to, or subsequent to, administration of a composition of the disclosure.

Compositions herein are administered either as a single dose or as part of a multidose regimen. Multidose regimens are often accompanied by ongoing monitoring of treatment efficacy, so as to assess impact on cell proliferation, disease cell count, tumor growth, or impact on disease or cancer symptom amelioration, among other outputs. Accordingly, treatment regimens are often adjusted in light of ongoing efficacy outcomes. Unfavorable treatment outcomes are addressed by, variously, increasing treatment dose, increasing treatment efficacy, or by changing or adding an additional target to a nuclease treatment regimen. Addition or alteration of accompanying treatment compositions is also contemplated and consistent with the disclosure herein.

As discussed above, some treatment regimens initially employ compositions directed to a known target sequence, switching to or supplementing with a target sequence identified de novo through tumor or other cancerous tissue sequencing when treatment outcome is not found to be leading to cancer clearance.

Table 5a shows illustrative gRNA sequences with a handle and T7 RNA polymerase promoter sequences and illustrative output information obtained using a Cas12a nuclease. Table 5b shows illustrative gRNA sequences with a handle, tracrRNA, and T7 RNA polymerase promoter sequences and illustrative output information obtained using a Cas9 nuclease. Table 6 and paragraph lists illustrative guide RNA sequences, which can be used to guide a polypeptide comprising a Cas12a nuclease domain (e.g., a chimeric polypeptide comprising a Cas12a nuclease) to a target nucleic acid associated with a cancer, for example, lung cancer or pancreatic cancer. Table 7 and paragraph [00305] lists illustrative guide RNA sequences, which can be used to guide a polypeptide comprising a Cas9 nuclease domain (e.g., a chimeric polypeptide comprising a Cas9 nuclease) to a target nucleic acid associated with a cancer, for example, lung cancer or pancreatic cancer. Variations from a reference sequence (e.g., wild type, healthy, or non-cancerous) are indicated in Table 5-Table 7 in underlined text.

Turning to the Figures, one sees the following.

At FIG. 1, CRISPR PLUS™ complexes are shown to be activated by gRNA-guided target site binding, which subsequently activate non-specific nuclease function. Degradation of DNA/RNA results in demolition of cancer cells. FIG. 1 shows a schematic of CRISPR PLUS™ complexes and a target nucleic acid (e.g., ds DNA and ss RNA). In each schematic, a portion of the guide RNA (gRNA) anneals to a target sequence, which is upstream of, and adjacent to, the protospacer adjacent motif (PAM).

At top, the action on double-stranded DNA is shown. A target sequence is identified, endonucleic cleavage resulting in a double-stranded break directed by binding to the target sequence is effected, and exonuclease activity amplifies the break to form a substantial lesion in the nucleic acid molecule leading to cell death.

At bottom, a complex identifies a target sequence in a ribonucleic acid molecule. The complex is activated and the RNA molecule is degraded.

At FIG. 2, the activation of the non-specific nuclease function of CRISPR PLUS™ is observed in cancer cells. A CRISPR PLUS™ complex is administered to an individual having a heterogeneous cell population. In this population, a mutation is identified as being associated with cell proliferation. Cells harboring this mutation are targeted for degradation. A ribonucleoprotein complex harboring a guide RNA that identifies the target mutation is assembled and administered to the individual. At left, one sees that cells lacking the mutation (no target sequence) are unaffected, because no target sequence is identified and no cell nucleic acid degradation occurs. At right, one sees that cells harboring the mutation (cancer specific sequence) are subjected to nucleic acid degradation. The guide RNA identifies the target mutation and directs endonuclease activity to cleave the nucleic acid. The non-specific nuclease function of CRISPR PLUS™ is activated specifically in cancer cell that contain a complementary target sequence of gRNA in the genome. Non-specific nuclease activity in cancer cells destroy cellular double stranded and single stranded DNA/RNA molecules, resulting in the induction of cell death.

At FIG. 3, the results of an in vitro cleavage assay are shown. A CRISPR/Cas12a ribonucleoprotein complex is assembled around a guide crRNA targeting human DHCR7. crRNA targets the dsDNA #1, comprising DHCR7, whereas it does not target the dsDNA #2, a nucleic acid lacking the target sequence. One sees that after 24 hours (second lane from the right), when CRISPR/Cas12a was activated by cutting its target DNA (dsDNA #1), it also collaterally degrades the dsDNA of non-target DNA (dsDNA #2). However, CRISPR/Cas12a complexes not previously activated by binding to a target dsDNA #1 show substantially less nonspecific degradation activity (center lane) as seen by the lack of degradation of dsDNA #2.

At FIG. 4, the results of an in vivo cleavage assay of the cytotoxic effect of CRISPR/Cas12a (FnCpf1-BPNLS) on Human cells are shown. At left is a graph showing cell viability on the y-axis, ranging from 0 to 3.5 in increments of 0.5, and treatment groups on the x-axis, including (from left to right) non-treated, Lipofectamine+Cpf1, Lipofectamine+sgRNA, Cpf1 (100 ng)+sgRNA, Cpf1 (100 ng)+sgRNA, Cpf1 (200 ng)+sgRNA), and Cpf1 (200 ng)+sgRNA). Within each treatment group are three bars, which from left to right show the 24 h time point, the 48 h timepoint, and the 72 h timepoint. At right is a graph showing cell viability on the y-axis, ranging from 0.000 to 3.500 in increments of 0.500, and treatment groups on the x-axis, including (from left to right) on-treated, Lipofectamine+Cpf1, Lipofectamine+sgRNA, Cpf1 (100 ng)+sgRNA, Cpf1 (100 ng)+sgRNA, Cpf1 (200 ng)+sgRNA), and Cpf1 (200 ng)+sgRNA). Within each treatment group are three bars, which from left to right show the 24 h time point, the 48 h timepoint, and the 72 h timepoint.

FIG. 5 shows a PX459 plasmid vector system for SpCas9 and sgRNA transfection with puromycin resistance. Shown from left to right are the U6 promoter, BbsI, BbsI, gRNA scaffold, CMV enhancer, chicken β-actin promoter, hybrid intron, 3xFLAG, SV40 NLS, Kozak sequence, Cas9, nucleoplasmin NLS, T2A, PuroR, bGH poly(A) signal, AAV2 ITR, f1 ori, AmpR promoter, AmpR, and ori.

FIG. 6A has a series of three images showing cells. From left to right, above each image, is a description of the treatment group, which respectively are pulse only, pSpCas9(BB)-2A-Puro (PX459) V2.0—EGFR_WT, and pSpCas9(BB)-2A-Puro (PX459) V2.0—EGFR_E2. The left most image with the pulse only group has many cells, indicating more live cells overall as compared to the right most image. The middle image with the pSpCas9(BB)-2A-Puro (PX459) V2.0—EGFR_WT group also has more live cells as compared to the right most image. The right image with the experimental group, pSpCas9(BB)-2A-Puro (PX459) V2.0—EGFR_E2, comprises less cells compared to the left and middle images, indicating more cell death.

FIG. 6B shows a graph, titled HCC827 cell number comparison (6 dpt) with the x-axis showing the three treatments including, from left to right, pulse only, EGFR_WT, and EGFR_E2. The y-axis shows the number of cells, ranging from 0 to 500000 in increments of 50000. Each treatment group additionally indicates error via error bars.

FIG. 7 shows two rows of microscope images of cells. The top left 3 images of the panel show many cells that have survived and are, from left to right, NT1, CCR5, and HPRT1. The top right image and all bottom images in the panel show images wherein most of the cells were found to be dead. The top right image shows the MT2 sample. The bottom row of the panel shows, from left to right, SMIM11, GNPDA2, SLC15A5, and KCNE2.

FIG. 8 shows a graph, which on the x-axis from left to right shows the different treatment groups comprising plasmids targeting gene loci. The groups from left to right are NT1, CCR5, HPRT1, MT2, SMIM11, GNPDA2, SLC15A5, and KCNE2. The y-axis shows the number of cells×10⁴ alive per well and ranges from 0 to 4 in increments of 0.5. Above each bar is the exact number of cells×10⁴ alive per well and are, from left to right, 3.36, 3.67, 3.52, 1.68, 0.89, 1.42, 1.86, and 0.76.

FIG. 9 shows a bar graph, which on the x-axis from left to right, shows the different treatment groups. These groups, from left to right, are Lipo, NT1, CCR5, HPRT1, MT2, GNPDA2, SLC15A5, and KCNE2. The y-axis shows the number of cells×10⁴ alive per well and ranges from 0.00 to 3.50 in increments of 0.50. Above each bar is the exact number of cells×10⁴ alive per well and are, from left to right, 3.02, 1.72, 2.33, 1.50, 0.43, 0.04, 0.02, and 0.04.

FIG. 10 shows on the top row, the NT1 sample, which from left to right show, a bright field image, a propidium iodide dead cell stain with a few red spots, and a NucBlue live cell stain reagent with many blue spots. The bottom row shows the GNPDA2 sample, which from left to right show, a bright field image, a propidium iodide dead cell stain with many red spots, and a NucBlue live cell stain reagent with many blue spots.

FIG. 11 shows a bar graph, which on the x-axis from left to right, shows the different treatment groups. These groups are, from left to right, CCR5, HPRT1, MT2, IRX1, and ADAMTS16. The y-axis shows the number of cells×10⁴ alive per well and ranges from 0 to 8 in increments of 1. At the top of each bar is the exact number of cells×10⁴ alive per well and are, from left to right, 7.33, 3.81, 3.17, 2.7, and 2.93. The title of the graph, shown at the top, is H1563 specific CNVs test.

FIG. 12A shows a bar graph, which on the x-axis, from left to right, shows the different time points tested and are 24 hr, 42 hr, and 72 hr. Within each timepoint, five groups were tested shown as 5 bars, which from left to right are pulse only, NT1, HPRT1-1, CCR5, and MT2. The y-axis shows a scale bar from 0.00 to 15000.00 in increments of 5000.00. The title of the graph, shown at the top, is A549_Celltiter glo

FIG. 12B shows a bar graph titled A549_counting. The x-axis, from left to right, shows the different treatment groups of pulse only, NT1, HPRT1, CCR5, and MT2. The y-axis shows the cell number (×10⁴) and ranges from 0 to 8 in increments of 2.

FIG. 13 shows microscope images showing cells after different treatments, which from left to right are pulse only, NT1, HPRT1, CCR5, and MT2. The NT1, HPRT1, and CCR5 images include arrows point to live cells.

FIG. 14 shows a bar graph titled A549 CellTiter Glo depicting a first trial, which on the x-axis, from left to right, shows the different time points tested and are 24 hr, 44 hr, and 51 hr. Within each timepoint, different groups were tested shown as bars, which from left to right are pulse only, pET21a, MT2, CD68, DACH2, HERC2P2, SHBG, and no pulse. The y-axis shows luminescence ranging from 0 to 7000 in increments of 1000.

FIG. 15A shows three graphs of the SK-BR-3 ERBB2 region whole genome sequence data from Nattestad, M. et al. (Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line. Genome Res. 28, 1126-1135 (2018).). Shown to the left of the three graphs is a scale bar, which at bottom indicates 10 Mbp and which at top indicates 0.1 Mbp. The top most graph shows Chromosome 17 and ERBB2 with no indels and shows the 37.84M to 37.89M region in increments of 0.01M. The middle graph shows the region scaling from 37M to 38.5M in increments of 0.5M and shows no small SCNAs. The bottom graph shows the region scaling from 20M to 80M in increments of 10M and shows many genes. An arrow indicates large SCNA. The y-axis on all three graphs ranges from 0 to 600 in increments of 100.

FIG. 15B shows a table, which from left to right shows chrom1, pos1, strand1, chrom2, post2, and strand2. Under chrom1 is 17, variant name, and 22790. Under pos1 is 26654135, variant_type, and DUP, under strand1 is “−”, split, and 37, under chrom2 is 17, size and 48414052, under pos2 is 75068187, CNV category, and matching, and under strand2 is “+”, category and solo.

FIG. 15C shows SK-BR-3 ERBB2 CNV junction sequence inferred from Nattestad et al. To the left, under the sequence, is chr17:75,058,188-75,068,187 and immediately to the right is chr17:26,654,135-26,664,134. Spanning these two regions is 60 bp of a junction region. Within the junction region are ERBB2 CTS, ERBB2 CT7, and ERBB2 CT6.

FIG. 16A shows a bar graph titled SKBR3_CellTiter Glo, which on the x-axis, from left to right, shows the different time points tested and are 24 hr, 42 hr, and 48 hr. Within each timepoint, different groups were tested shown as bars, which from left to right are pulse only, NT1, MT2, ERBB2, KRT16, and no pulse. The axis shows luminescence ranging from 0 to 4000 in increments of 1000.

FIG. 16B shows a bar graph, which is the raw data for the graph in FIG. 16A. The x-axis, from left to right, shows the different time points tested and are 24 hr, 42 hr, and 48 hr. Within each timepoint, different groups were tested shown as bars, which from left to right are pulse only, NT1, HPRT1, MT2, ERBB2, KRT16, and no pulse. The axis shows values ranging from 0 to 4000 in increments of 500.

FIG. 17 shows a bar graph, which on the x-axis, from left to right, shows the different groups tested including pulse only, NT1, HPRT1, MT2, ERBB2, and KRT16. The y-axis shows cell number (×10{circumflex over ( )}5) ranging from 0 to 15 in increments of 5.

FIG. 18A shows HPV and HeLa cell integration data from Adey et al. Purple is HeLa (shown in the HepB panel and shown at top in the HepA panel) and green is HPV (shown at bottom in the HepA panel). The strength of the signal is shown by the color of the shading. The key at the top left shows the legend, which shows purple shading on the top row and green shading on the bottom row. The top row of the legend shows from left to right, 4x, 34x, and 13x. The bottom row of the legend shows, from left to right, 30x, 12x, and 3x.

FIG. 18B shows HPV sequence data in HeLa cell inferred from Meissner et al, Landry et al, Adey et al., and Liu et al. Shown in the diagram is the human chromosome. From left to right, the following regions are shown: 1X, 3X, and 6X. Repeat numbers are marked as X and only opened sequences are marked as arrow. The text below the diagram shows annotations of specific regions, which, from left to right, are Human 2a, HPV7, HPV4, Human 1, Human 3, HPV 6, HPV4, and Human 1.

FIG. 18C shows a gel of the PCR product of the repeat region between HPV sequences in HeLa cells, based on the references noted above in FIG. 18B, which shows a ladder in the right most lane. Arrows point to 7 kb and 2 kb.

FIG. 19A shows a graph titled cell counting, which on the x-axis, from left to right, shows different groups tested incluing CCR5, MT2, and PRDM9. The y-axis shows live cell numbered ranging from 0 to 20000 in increments of 2000.

FIG. 19B shows a graph titled cell counting, which on the x-axis, from left to right, shows different groups tested including CCR5, MT2, PRDM9, and HPV_1. The y-axis shows live cell numbered ranging from 0 to 35000 in increments of 5000.

FIG. 20A shows a graph titled Cell Titer Glo and shows on the x-axis, from left to right, different timepoints that were tested including 24 h, 48 h, and 72 h. Within each timepoint, different groups were tested, which from left to right are pulse only (GFP), CCR5, MT2, HPV_1, and PRDM9. The y-axis shows luminescence signal ranging from 0 to 6000 in increments of 1000.

FIG. 20B shows a graph titled Cell Titer Glo. The y-axis shows the relative luminescence signal, ranging from 0 to 100 in increments of 20, and the x-axis shows the different groups tested, which are (from left to right) NT1, NT2, NT3, CCR5, MT2, and HPV_1.

FIG. 21 shows a graph, which shows different groups tested on the x-axis. Shown on the x-axis, from left to right, are NT1, CCR5, HPRT1, MT2, TRAPPC, LINC00536, TRPS1, and CDK8. The y-axis shows cell number (×10{circumflex over ( )}4 cells/well) ranging from 0.00 to 60.00 in increments of 10.00.

FIG. 22 shows a graph titled cross check (1299 targets in 1563). The x-axis shows different groups tested, which from left to right are CCR5, SMIM11, GNPDA2, SLC15A5, and KCNE2. The y-axis shows the number of cells (×10{circumflex over ( )}4 cells alive per well) ranging from 0 to 14 in increments of 2. In the bar displayed for each group is the exact number of cells (×10{circumflex over ( )}4) alive per well, which from left to right are 7.33, 10.7, 13, 12, and 3.7.

FIG. 23 shows microscope images of cells. The top row shows, from left to right, SMIM11 H1563 and GNPDA2 H1563. The bottom right shows, from left to right, SLC15A5 H1563 and KCNE2 H1563.

FIG. 24 shows a graph, which on the x-axis shows, from left to right, pulse only, NT1, CCR5, KCNE2, GNPDA2, SMIM11, and SLC15A5. The y-axis shows luminescence ranging from 0 to 14000 in increments of 2000. The graph is titled H1299_AnnV+.

FIG. 25 shows at top microscope images, which from left to right, are pulse only, PX459-EGFR_WT, PX459-CCR5, PX459-EGFR_E2, PX459-RecJ-CCR5, and PX459-RecJ-EGFR_E2. At the bottom is a graph, which from left to right, shows pulse only, PX459-EGFR_WT, PX459-CCR5, PX459-EGFR_E2, PX459-RecJ-CCR5, and PX459-Red-EGFR_E2. The y-axis shows live cell signal (luminescence) ranging from 0 to 40000 in increments of 5000.

FIG. 26 shows a graph, which on the x-axis shows, from left to right, pulse only, Cas9-GFP only, sgRNA only, and EGFR_E2 RNP. The y-axis shows the cell number change % ranging from −80 to 20 in increments of 20. The title of the graph, shown at the top, is Live cell signal 24 h after electroporation.

FIG. 27 shows a graph of H1563 cell viability assay at the 48 hr time point. The x-axis, from left to right, shows pulse only, sgRNA only, and MT2 RNP. The y-axis shows the live cell signal (luminescence) ranging from 0 to 150 in increments of 50. The title, shown at the top of the graph, is H1563 cell viability assay (48 hpt).

FIG. 28 at top shows bright field images of cells, which from left to right are mock (Cas9GFP only), Mock (sgRNA only), single target (CCR5 RNP), multi-target (GNPDA2 RNP), and multi-target (SMIM11 RNP). The bottom left is a graph, which from left to right on the x-axis shows mock (Cas9GFP only), Mock (sgRNA only), single target (CCR5 RNP), multi-target (GNPDA2 RNP), and multi-target (SMIM11 RNP) and which on the y-axis shows the live cell signal (luminescence) from 0 to 25000 in increments of 5000. The bottom right is a graph, which from left to right on the x-axis shows mock (Cas9GFP only), Mock (sgRNA only), single target (CCR5 RNP), and multi-target (MT2). The y-axis shows the live cell signal (luminescence) from 0 to 20000 in increments of 5000.

FIG. 29 shows a bar graph, which shows on the x-axis (from left to right) the treatment groups of pulse only, NT1 (non-target), MT2, CDK8, LINC00536, TRPS1, and TRAPPC9. The y-axis shows luminescence and ranges from 0 to 1400 in increments of 200.

FIG. 30 shows the target sequence of AsCpf1_crRNA and Cpf 1 crRNA binding site WT. To the left of the target binding region is the end of the U6 promoter region and on the bottom row of sequence is the start of the CMV enhancer region.

FIG. 31 shows, at top, bright field images of cells in different treatment groups, which are, from left to right, Cas12a-EGFR WT, Cas12a-CCR5, and Cas12a-EGFR E2. At bottom is a bar graph, which on the x-axis shows the different treatment groups of, from left to right, Cas12a-EGFR WT, Cas12a-CCR5, and Cas12a-EGFR E2. The y-axis shows the live cell signal in luminescence ranging from 0 to 4500 in increments of 500.

Numbered Embodiments

The following embodiments recite permutations of combinations of features disclosed herein. In some cases, permutations of combinations of features disclosed herein are non-limiting. In other cases permutations of combinations of features disclosed herein are limiting. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. Some methods enumerated herein include up to and all of the following elements a, b, and c. The methods may further include additional elements. 1. A composition to induce cell death, the composition comprising: a) a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and b) a guide nucleic acid comprising a sequence complementary to a target nucleic acid in a cell, wherein the target nucleic acid is associated with a disorder. 2. The composition of embodiment 1, wherein the disorder is cancer. 3. The composition of embodiment 2, wherein the cancer is lung cancer or pancreatic cancer. 4. The composition of embodiment 1, wherein the target nucleic acid comprises RNA or DNA. 5. The composition of embodiment 1, wherein the target nucleic acid comprises a sequence associated with the disorder. 6. The composition of embodiment 1, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 7. The composition of embodiment 5, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 8. The composition of embodiment 1, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer, a translocation, or a sequence associated with cancer progression. 9. The composition of embodiment 1, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, and HER2. 10. The composition of embodiment 1, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 11. The composition of embodiment 10, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 12. The composition of embodiment 1, wherein the first domain comprises a Cas12a (or Cpf1) domain, a Cas12a domain, a Cas12b domain, a Cas12c domain, a Cas12d domain, a Cas12e domain, a Cas12f domain, a Cas12g domain, a Cas12h domain, a Cas12i domain, a Cas13a domain, a Cas13b domain, a Cas14 domain, or a Cas9 domain. 13. The composition of embodiment 1, wherein the first domain comprises a Cas12a (or Cpf1) domain. 14. The composition of embodiment 1, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 15. The composition of embodiment 1, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 16. The composition of embodiment 1, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 17. The composition of embodiment 1, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 18. The composition of embodiment 17, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the first domain. 19. The composition of embodiment 1, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 20. The composition of embodiment 1, wherein the chimeric polypeptide generates a 3′ OH overhang. 21. The composition of embodiment 1, wherein the chimeric polypeptide exposes a recessed 3′ OH. 22. The composition of embodiment 1, wherein the second domain comprises an enzyme having cleaved end resection activity. 23. The composition of embodiment 1, wherein the second domain comprises mung bean nuclease. 24. The composition of embodiment 1, wherein the chimeric polypeptide and the guide nucleic acid are present as a ribonucleoprotein complex in the composition. 25. The composition of embodiment 1, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 26. The composition of embodiment 1, wherein the composition is formulated for oral administration. 27. The composition of embodiment 1, wherein the composition is formulated as a pill, a tablet, or a capsule. 28. The composition of embodiment 1, wherein the composition is formulated with an enteric coating. 29. The composition of embodiment 1, wherein the composition is formulated for topical administration. 30. The composition of embodiment 1, wherein the composition is formulated for parenteral administration. 31. The composition of embodiment 1, wherein the composition is formulated for intrathecal administration. 32. The composition of embodiment 1, wherein the composition further comprises an agent to facilitate entry of the composition into a cell comprising the target nucleic acid. 33. The composition of embodiment 1, wherein the composition further comprises a buffer. 34. A method for inducing cell death, the method comprising contacting a target nucleic acid in a cell with a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity, thereby inducing death of the cell. 35. The method of embodiment 34, wherein the cell is a cancer cell. 36. The method of embodiment 34, wherein the target nucleic acid comprises a sequence associated with a disorder. 37. The method of embodiment 36, wherein the disorder is cancer. 38. The method of embodiment 36, wherein the disorder is lung cancer or pancreatic cancer. 39. The method of embodiment 34, wherein the target nucleic acid comprises RNA or DNA. 40. The method of embodiment 34, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 41. The method of embodiment 34, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 42. The method of embodiment 34, wherein the target nucleic acid comprises a translocation, a single nucleotide polymorphism specific to a cancer, or a sequence associated with cancer progression. 43. The method of embodiment 34, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 44. The method of embodiment 34, further comprising contacting the target nucleic acid with a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 45. The method of embodiment 44, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 46. The method of embodiment 34, further comprising site-specifically cleaving the target nucleic acid by the chimeric polypeptide. 47. The method of embodiment 46, further comprising non-specifically cleaving nucleic acids in the cell. 48. The method of embodiment 34, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 49. The method of embodiment 58, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 50. The method of embodiment 34, wherein the first domain comprises a Cas12a (or Cpf1) domain, a Cas12a domain, a Cas12b domain, a Cas12c domain, a Cas12d domain, a Cas12e domain, a Cas12f domain, a Cas12g domain, a Cas12h domain, a Cas12i domain, a Cas13a domain, a Cas13b domain, a Cas14 domain, or a Cas9 domain. 51. The method of embodiment 34, wherein the first domain comprises a Cas12a (or Cpf1) domain. 52. The method of embodiment 34, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 53. The composition of embodiment 34, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 54. The composition of embodiment 34, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 55. The method of embodiment 34, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 56. The method of embodiment 55, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acid by the first domain. 57. The method of embodiment 34, wherein the second domain comprises a RecE domain, a RecJ domain, a T4 domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 58. The method of embodiment 34, wherein the chimeric polypeptide generates a 3′ OH overhang. 59. The method of embodiment 34, wherein the chimeric polypeptide exposes a recessed 3′ OH. 60. The method of embodiment 34, wherein the second domain comprises an enzyme having cleaved end resection activity. 61. The method of embodiment 34, wherein the second domain comprises mung bean nuclease. 62. The method of embodiment 34, wherein the chimeric polypeptide is delivered to the cell in a ribonucleoprotein complex comprising the chimeric polypeptide and a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 63. The method of embodiment 34, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acid by the chimeric polypeptide. 64. The method of embodiment 63, wherein the terminating is performed by administering to the subject at least one of: an antibody specific to the chimeric polypeptide or ZnSO4. 65. A composition comprising: a) a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and b) a guide nucleic acid comprising: a first section comprising at least 5 consecutive bases comprising complete complementarity to a portion of a first chromosome of a healthy cell, and a second section comprising at least 5 consecutive bases comprising complete complementarity to a portion of a second chromosome of a healthy cell. 66. The composition of embodiment 65, wherein the first section comprises complementarity to a portion of BCR gene and the second section comprises complementarity to a portion of ABL gene. 67. The composition of embodiment 65, wherein the first section comprises complementarity to a portion of TEL gene and the second section comprises complementarity to a portion of AML gene. 68. The composition of embodiment 65, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 69. The composition of embodiment 65, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of a target nucleic acid by the chimeric polypeptide. 70. The composition of embodiment 65, wherein the first domain comprises a Cas9 domain. 71. The composition of embodiment 65, wherein the first domain comprises a Cas12a (or Cpf1) domain. 72. The composition of embodiment 65, wherein the first domain comprises a Cas13a or Cas13b domain. 73. The composition of embodiment 65, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 74. The composition of embodiment 73, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of a target nucleic acid by the first domain. 75. The composition of embodiment 65, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex1 domain. 76. The composition of embodiment 65, wherein the chimeric polypeptide generates a 3′ OH overhang. 77. The composition of embodiment 65, wherein the chimeric polypeptide exposes a recessed 3′ OH. 78. The composition of embodiment 65, wherein the second domain comprises a domain having cleaved end resection activity. 79. The composition of embodiment 65, wherein the second domain comprises mung bean nuclease. 80. The composition of embodiment 65, wherein the chimeric polypeptide and the guide nucleic acid are present as a ribonucleoprotein complex in the composition. 81. The composition of embodiment 65, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 82. The composition of embodiment 65, wherein the composition is formulated for oral administration. 83. The composition of embodiment 65, wherein the composition is formulated as a pill, a tablet, or a capsule. 84. The composition of embodiment 65, wherein the composition is formulated with an enteric coating. 85. The composition of embodiment 65, wherein the composition is formulated for topical administration. 86. The composition of embodiment 65, wherein the composition is formulated for parenteral administration. 87. The composition of embodiment 65, wherein the composition is formulated for intrathecal administration. 88. The composition of embodiment 65, wherein the composition further comprises an agent to facilitate entry of the composition into a cell comprising the target nucleic acid. 89. The composition of embodiment 65, wherein the composition further comprises a buffer. 90. A method for ameliorating a symptom in a subject having cancer, the method comprising contacting a target nucleic acid in the subject with a composition of any one of embodiments 65 to 89. 91. The method of embodiment 90, wherein the target nucleic acid is in a region of chromosomal translocation involving the portion of the first chromosome and the portion of the second chromosome. 92. The method of embodiment 90, wherein the target nucleic acid hybridizes with the first section and the second section of the guide nucleic acid. 93. The method of embodiment 90, further comprising administering the composition to the subject, wherein chimeric polypeptide and the guide nucleic acid are in a ribonucleoprotein complex in the composition. 94. The method of embodiment 90, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acid by the chimeric polypeptide. 95. The method of embodiment 90, wherein the terminating is performed by administering to the subject an antibody specific to the chimeric polypeptide. 96. The method of embodiment 90, wherein the terminating is performed by administering to the subject ZnSO4. 97. A composition for targeting a pathogen, the composition comprising: a) a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and b) a guide nucleic acid comprising a sequence complementary to a target nucleic acid of a pathogen. 98. The composition of embodiment 97, wherein the target nucleic acid comprises RNA. 99. The composition of embodiment 97, wherein the target nucleic acid comprises DNA. 100. The composition of embodiment 97, wherein the pathogen is a virus. 101. The composition of embodiment 97, wherein the pathogen is a bacterium. 102. The composition of embodiment 97, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 103. The composition of embodiment 102, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 104. The composition of embodiment 97, wherein the first domain comprises a Cas12a (or Cpf1) domain. 105. The composition of embodiment 97, wherein the first domain comprises a Cas13a or Cas13b domain. 106. The composition of embodiment 97, wherein the first domain comprises a Cas9 domain. 107. The composition of embodiment 97, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 108. The composition of embodiment 107, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acid by the first domain. 109. The composition of embodiment 97, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 110. The composition of embodiment 97, wherein the chimeric polypeptide generates a 3′ OH overhang. 111. The composition of embodiment 97, wherein the chimeric polypeptide exposes a recessed 3′ OH. 112. The composition of embodiment 97, wherein the second domain comprises an enzyme having cleaved end resection activity. 113. The composition of embodiment 97, wherein the second domain comprises mung bean nuclease. 114. The composition of embodiment 97, wherein the chimeric polypeptide and the guide nucleic acid are present as a ribonucleoprotein complex in the composition. 115. The composition of embodiment 97, wherein the guide nucleic acid does not bind to a host nucleic acid. 116. The composition of embodiment 97, wherein the composition is formulated for oral administration. 117. The composition of embodiment 97, wherein the composition is formulated as a pill, a tablet, or a capsule. 118. The composition of embodiment 97, wherein the composition is formulated with an enteric coating. 119. The composition of embodiment 97, wherein the composition is formulated for topical administration. 120. The composition of embodiment 97, wherein the composition is formulated for parenteral administration. 121. The composition of embodiment 97, wherein the composition is formulated for intrathecal administration. 122. The composition of embodiment 97, wherein the composition further comprises an agent to facilitate entry of the composition into a cell comprising the target nucleic acid. 123. The composition of embodiment 97, wherein the composition further comprises a buffer. 124. A method for targeting a pathogen in a subject, the method comprising contacting a target nucleic acid of a pathogen in the subject with a composition of any one of embodiments 97 to 123. 125. The method of embodiment 124, wherein the chimeric polypeptide cleaves the target nucleic acid. 126. The method of embodiment 124, further comprising administering the composition to the subject, wherein chimeric polypeptide and the guide nucleic acid are in a ribonucleoprotein complex in the composition. 127. The method of embodiment 124, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acid by the chimeric polypeptide. 128. The method of embodiment 124, wherein the terminating is performed by administering to the subject an antibody specific to the chimeric polypeptide. 129. The method of embodiment 124, wherein the terminating is performed by administering to the subject ZnSO4. 130. A method for ameliorating a symptom in a subject having a disorder, the method comprising administering to the subject a ribonucleoprotein complex comprising: (i) a polypeptide comprising sequence-specific endonuclease activity; and (ii) a guide nucleic acid comprising a sequence complementary to a target nucleic acid in the subject. 131. The method of embodiment 130, wherein the disorder is cancer. 132. The method of embodiment 130, wherein the disorder is lung cancer or pancreatic cancer. 133. The method of embodiment 130, wherein the target nucleic acid comprises a sequence associated with the disorder. 134. The method of embodiment 130, wherein the target nucleic acid comprises RNA. 135. The method of embodiment 130, wherein the target nucleic acid comprises DNA. 136. The method of embodiment 130, wherein the target nucleic acid is in a cancer cell of the subject. 137. The method of embodiment 130, wherein the target nucleic acid is absent in a healthy cell of the subject. 138. The method of embodiment 130, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 139. The method of embodiment 138, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 140. The method of embodiment 139, wherein the target nucleic acid comprises a translocation. 141. The method of embodiment 130, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer. 142. The method of embodiment 130, wherein the target nucleic acid comprises a sequence associated with cancer progression. 143. The method of embodiment 130, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 144. The method of embodiment 130, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 145. The method of embodiment 130, further comprising site-specifically cleaving the target nucleic acid by the polypeptide. 146. The method of embodiment 130, further comprising non-specifically cleaving other nucleic acids in a cell comprising the target nucleic acid by the polypeptide. 147. The method of embodiment 130, wherein the polypeptide further comprises inducible non-specific nuclease activity. 148. The method of embodiment 147, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the polypeptide. 149. The method of embodiment 130, wherein the polypeptide comprises a Cas12a (or Cpf1) domain. 150. The method of embodiment 130, wherein the polypeptide comprises a Cas13a or Cas13b domain. 151. The method of embodiment 130, wherein the polypeptide comprises a Cas9 domain. 152. The method of embodiment 130, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 153. The composition of embodiment 130, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 154. The composition of embodiment 130, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 155. The method of embodiment 130, wherein the polypeptide is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 156. The method of embodiment 155, wherein the polypeptide is a chimeric polypeptide comprising a first domain comprising the sequence-specific endonuclease activity and a second domain comprising exonuclease activity. 157. The method of embodiment 156, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 158. The method of embodiment 156, wherein the chimeric polypeptide generates a 3′ OH overhang. 159. The method of embodiment 156, wherein the chimeric polypeptide exposes a recessed 3′ OH. 160. The method of embodiment 156, wherein the second domain comprises an enzyme having cleaved end resection activity. 161. The method of embodiment 156, wherein the second domain comprises mung bean nuclease. 162. The method of embodiment 130, further comprising terminating activity of the polypeptide in the subject after cleavage of the target nucleic acid by the polypeptide. 163. The method of embodiment 130, wherein the terminating is performed by administering to the subject an antibody specific to the polypeptide. 164. The method of embodiment 130, wherein the terminating is performed by administering to the subject ZnSO4. 165. A method for ameliorating a symptom in a subject having cancer, the method comprising a) administering to the subject a therapy selected from the group consisting of: chemotherapy, radiation therapy, immunotherapy, hormone therapy, and any combination thereof; and b) contacting a target nucleic acid in the subject with a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity. 166. The method of embodiment 165, wherein the therapy is immunotherapy. 167. The method of embodiment 166, wherein the immunotherapy comprises CAR T-cell therapy. 168. The method of embodiment 166, wherein the immunotherapy comprises administering trastuzumab 169. The method of embodiment 165, wherein the therapy is chemotherapy. 170. The method of embodiment 169, wherein the chemotherapy comprises administering imatinib mesylate. 171. The method of embodiment 165, wherein the combination of a) and b) provides an enhanced therapeutic effect compared to the therapeutic effect of a) or b) alone. 172. The method of embodiment 165, wherein the target nucleic acid is in a cancer cell of the subject. 173. The method of embodiment 165, wherein the target nucleic acid is absent in a healthy cell of the subject. 174. The method of embodiment 165, wherein the target nucleic acid comprises a sequence associated with the cancer. 175. The method of embodiment 165, wherein the target nucleic acid comprises RNA. 176. The method of embodiment 165, wherein the target nucleic acid comprises DNA. 177. The method of embodiment 165, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 178. The method of embodiment 177, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 179. The method of embodiment 177, wherein the target nucleic acid comprises a translocation. 180. The method of embodiment 165, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer. 181. The method of embodiment 165, wherein the target nucleic acid comprises a sequence associated with cancer progression. 182. The method of embodiment 165, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 183. The method of embodiment 165, further comprising contacting the target nucleic acid with a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 184. The method of embodiment 183, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 185. The method of embodiment 183, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 186. The composition of embodiment 183, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 187. The composition of embodiment 183, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 188. The method of embodiment 165, further comprising site-specifically cleaving the target nucleic acid by the chimeric polypeptide. 189. The method of embodiment 165, further comprising non-specifically cleaving other nucleic acids in a cell comprising the target nucleic acids by the chimeric polypeptide. 190. The method of embodiment 165, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 191. The method of embodiment 165, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 192. The method of embodiment 165, wherein the first domain comprises a Cas12a (or Cpf1) domain. 193. The method of embodiment 165, wherein the first domain comprises a Cas13a or Cas13b domain. 194. The method of embodiment 165, wherein the first domain comprises a Cas9 domain. 195. The method of embodiment 165, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 196. The method of embodiment 195, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acid by the first domain. 197. The method of embodiment 165, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 198. The method of embodiment 165, wherein the chimeric polypeptide generates a 3′ OH overhang. 199. The method of embodiment 165, wherein the chimeric polypeptide exposes a recessed 3′ OH. 200. The method of embodiment 165, wherein the second domain comprises an enzyme having cleaved end resection activity. 201. The method of embodiment 165, wherein the second domain comprises mung bean nuclease. 202. The method of embodiment 165, wherein the chimeric polypeptide is delivered to the cell in a ribonucleoprotein complex comprising the chimeric polypeptide and a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 203. The method of embodiment 165, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acid by the chimeric polypeptide. 204. The method of embodiment 203, wherein the terminating is performed by administering to the subject an antibody specific to the chimeric polypeptide. 205. The method of embodiment 203, wherein the terminating is performed by administering to the subject ZnSO4. 206. A method for ameliorating a symptom in a subject having cancer, the method comprising a) quantifying a first health condition of the subject at a first time point; b) administering to the subject a therapy selected from the group consisting of: chemotherapy, radiation therapy, immunotherapy, hormone therapy, and any combination thereof; c) quantifying a second health condition of the subject at a second time point after the therapy; d) contacting a target nucleic acid in the subject with a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and e) quantifying a third health condition of the subject at a third time point after the contacting. 207. The method of embodiment 206, wherein the quantifying the first health condition, the second health condition, or the third health condition comprises use of one or more of: biopsy, imaging test, genetic test, tumor molecular profiling, laboratory test, or nucleic acid sequencing. 208. The method of embodiment 206, wherein the therapy is immunotherapy. 209. The method of embodiment 208, wherein the immunotherapy comprises CAR T-cell therapy. 210. The method of embodiment 208, wherein the immunotherapy comprises administering trastuzumab 211. The method of embodiment 206, wherein the therapy is chemotherapy. 212. The method of embodiment 211, wherein the chemotherapy comprises administering imatinib mesylate. 213. The method of embodiment 206, wherein the third health condition of the subject is better than the first health condition and the second health condition. 214. The method of embodiment 206, wherein the target nucleic acid is in a cancer cell of the subject. 215. The method of embodiment 206, wherein the target nucleic acid is absent in a healthy cell of the subject. 216. The method of embodiment 206, wherein the target nucleic acid comprises a sequence associated with the cancer. 217. The method of embodiment 206, wherein the target nucleic acid comprises RNA. 218. The method of embodiment 206, wherein the target nucleic acid comprises DNA. 219. The method of embodiment 206, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 220. The method of embodiment 219, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 221. The method of embodiment 219, wherein the target nucleic acid comprises a translocation. 222. The method of embodiment 206, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer. 223. The method of embodiment 206, wherein the target nucleic acid comprises a sequence associated with cancer progression. 224. The method of embodiment 206, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 225. The method of embodiment 206, further comprising contacting the target nucleic acid with a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 226. The method of embodiment 225, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 227. The method of embodiment 225, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 228. The composition of embodiment 225, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 229. The composition of embodiment 225, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 230. The method of embodiment 206, further comprising site-specifically cleaving the target nucleic acid by the chimeric polypeptide. 231. The method of embodiment 206, further comprising non-specifically cleaving other nucleic acids in a cell comprising the target nucleic acids by the chimeric polypeptide. 232. The method of embodiment 206, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 233. The method of embodiment 232, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 234. The method of embodiment 206, wherein the first domain comprises a Cas12a (or Cpf1) domain. 235. The method of embodiment 206, wherein the first domain comprises a Cas13a or Cas13b domain. 236. The method of embodiment 206, wherein the first domain comprises a Cas9 domain. 237. The method of embodiment 206, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 238. The method of embodiment 237, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acid by the first domain. 239. The method of embodiment 206, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 240. The method of embodiment 206, wherein the chimeric polypeptide generates a 3′ OH overhang. 241. The method of embodiment 206, wherein the chimeric polypeptide exposes a recessed 3′ OH. 242. The method of embodiment 206, wherein the second domain comprises an enzyme having cleaved end resection activity. 243. The method of embodiment 206, wherein the second domain comprises mung bean nuclease. 244. The method of embodiment 206, wherein the chimeric polypeptide is delivered to the cell in a ribonucleoprotein complex comprising the chimeric polypeptide and a guide nucleic acid comprising a sequence complementary to the target nucleic acid. 245. The method of embodiment 206, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acid by the chimeric polypeptide. 246. The method of embodiment 245, wherein the terminating is performed by administering to the subject an antibody specific to the chimeric polypeptide. 247. The method of embodiment 245, wherein the terminating is performed by administering to the subject ZnSO4. 248. A method for terminating activity of a polypeptide in a subject, the method comprising: a) cleaving a target nucleic acid in a subject with a polypeptide comprising sequence-specific endonuclease activity; and b) terminating activity of the polypeptide in the subject. 249. The method of embodiment 248, wherein the terminating comprises administering to the subject an antibody specific to the polypeptide. 250. The method of embodiment 248, wherein the terminating comprises administering to the subject ZnSO4. 251. The method of embodiment 248, wherein the subject has a disorder. 252. The method of embodiment 251, wherein the disorder is cancer. 253. The method of embodiment 251, wherein the disorder is lung cancer or pancreatic cancer. 254. The method of embodiment 251, wherein the target nucleic acid comprises a sequence associated with the disorder. 255. The method of embodiment 248, wherein the target nucleic acid comprises RNA. 256. The method of embodiment 248, wherein the target nucleic acid comprises DNA. 257. The method of embodiment 248, wherein the target nucleic acid is in a cancer cell of the subject. 258. The method of embodiment 248, wherein the target nucleic acid is absent in a healthy cell of the subject. 259. The method of embodiment 248, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 260. The method of embodiment 248, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 261. The method of embodiment 260, wherein the target nucleic acid comprises a translocation. 262. The method of embodiment 248, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer. 263. The method of embodiment 248, wherein the target nucleic acid comprises a sequence associated with cancer progression. 264. The method of embodiment 248, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 265. The method of embodiment 248, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 266. The method of embodiment 248, further comprising site-specifically cleaving the target nucleic acid by the polypeptide. 267. The method of embodiment 248, further comprising non-specifically cleaving other nucleic acids in a cell comprising the target nucleic acid by the polypeptide. 268. The method of embodiment 248, wherein the polypeptide further comprises inducible non-specific nuclease activity. 269. The method of embodiment 248, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the polypeptide. 270. The method of embodiment 248, wherein the polypeptide comprises a Cas12a (or Cpf1) domain. 271. The method of embodiment 248, wherein the polypeptide comprises a Cas13a or Cas13b domain. 272. The method of embodiment 248, wherein the polypeptide comprises a Cas9 domain. 273. The method of embodiment 248, wherein the polypeptide is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 274. The method of embodiment 248, wherein the polypeptide is a chimeric polypeptide comprising a first domain comprising the sequence-specific endonuclease activity and a second domain comprising exonuclease activity. 275. The method of embodiment 248, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 276. The method of embodiment 248, wherein the chimeric polypeptide generates a 3′ OH overhang. 277. The method of embodiment 248, wherein the chimeric polypeptide exposes a recessed 3′ OH. 278. The method of embodiment 248, wherein the second domain comprises an enzyme having cleaved end resection activity. 279. The method of embodiment 248, wherein the second domain comprises mung bean nuclease. 280. A composition for targeting two or more nucleic acids, the composition comprising a) a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and b) two or more guide nucleic acids, wherein each guide nucleic acid comprises a sequence complementary to a different target nucleic acid in a cell. 281. The composition of embodiment 280, wherein the target nucleic acids comprise RNA. 282. The composition of embodiment 280, wherein the target nucleic acids comprise DNA. 283. The composition of embodiment 280, wherein the target nucleic acids comprise a sequence associated with the disorder. 284. The composition of embodiment 283, wherein the disorder is cancer. 285. The composition of embodiment 283, wherein the disorder is lung cancer or pancreatic cancer. 286. The composition of embodiment 283, wherein the guide nucleic acids comprise a sequence selected from the group consisting of the sequences in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 287. The composition of embodiment 283, wherein the guide nucleic acid comprises a first portion and a second portion, wherein the first portion comprises at least about 85% sequence identity to: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat (SEQ ID NO: 65), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer. 288. The composition of embodiment 283, wherein the guide nucleic acid comprises a first portion, a second portion, and a third portion, wherein the first portion comprises at least about 85% sequence identity to the sequence: attctaatacgactcactatagg (SEQ ID NO: 3300), wherein the second portion comprises a sequence complementary to a region of a gene associated with cancer, and wherein the third portion comprises at least about 85% sequence identity to the sequence: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 66). 289. The composition of embodiment 280, wherein the cell is a cancer cell. 290. The composition of embodiment 280, wherein the target nucleic acids are within regions comprising a chromosomal abnormality. 291. The composition of embodiment 290, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 292. The composition of embodiment 280, wherein the target nucleic acids comprise a single nucleotide polymorphism specific to a cancer. 293. The composition of embodiment 291, wherein the target nucleic acids comprise a translocation. 294. The composition of embodiment 280, wherein the target nucleic acids comprise a sequence associated with cancer progression. 295. The composition of embodiment 280, wherein the target nucleic acids comprise a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 296. The composition of embodiment 280, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 297. The composition of embodiment 296, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acids by the chimeric polypeptide. 298. The composition of embodiment 280, wherein the first domain comprises a Cas12a (or Cpf1) domain. 299. The composition of embodiment 280, wherein the first domain comprises a Cas13a or Cas13b domain. 300. The composition of embodiment 280, wherein the first domain comprises a Cas9 domain. 301. The composition of embodiment 280, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 302. The composition of embodiment 301, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acids by the first domain. 303. The composition of embodiment 280, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 304. The composition of embodiment 280, wherein the chimeric polypeptide generates a 3′ OH overhang. 305. The composition of embodiment 280, wherein the chimeric polypeptide exposes a recessed 3′ OH. 306. The composition of embodiment 280, wherein the second domain comprises an enzyme having cleaved end resection activity. 307. The composition of embodiment 280, wherein the second domain comprises mung bean nuclease. 308. The composition of embodiment 280, wherein the guide nucleic acids do not bind to nucleic acids in a healthy cell. 309. The composition of embodiment 280, wherein the composition comprises one or more ribonucleoprotein complexes, wherein each ribonucleoprotein complex comprises the chimeric polypeptide and a guide nucleic acid from the two or more guide nucleic acids. 310. The composition of embodiment 280, wherein the composition is formulated for oral administration. 311. The composition of embodiment 280, wherein the composition is formulated as a pill, a tablet, or a capsule. 312. The composition of embodiment 280, wherein the composition is formulated with an enteric coating. 313. The composition of embodiment 280, wherein the composition is formulated for topical administration. 314. The composition of embodiment 280, wherein the composition is formulated for parenteral administration. 315. The composition of embodiment 280, wherein the composition is formulated for intrathecal administration. 316. The composition of embodiment 280, wherein the composition further comprises an agent to facilitate entry of the composition into a cell comprising the target nucleic acid. 317. The composition of embodiment 280, wherein the composition further comprises a buffer. 318. A method for ameliorating a symptom of a subject having a disorder, the method comprising contacting target nucleic acids in the subject with a composition of any one of embodiments 280 to 317. 319. The method of embodiment 318, wherein the disorder is cancer. 320. The method of embodiment 318, wherein the disorder is lung cancer or pancreatic cancer. 321. The method of embodiment 318, further comprising cleaving the target nucleic acids by the chimeric polypeptide. 322. The method of embodiment 318, further comprising inducing death of the cell. 323. The method of embodiment 318, further comprising terminating activity of the chimeric polypeptide in the subject after cleavage of the target nucleic acids by the chimeric polypeptide. 324. The method of embodiment 323, wherein the terminating is performed by administering to the subject an antibody specific to the chimeric polypeptide. 325. The method of embodiment 323, wherein the terminating is performed by administering to the subject ZnSO4. 326. A method for ameliorating a symptom in a subject having cancer, the method comprising: a) obtaining a biological sample of the subject; b) determining sequence information of nucleic acids in the biological sample; c) identifying a target nucleic acid from the sequence information, wherein the target nucleic acid is associated with the cancer; d) administering a composition comprising a ribonucleoprotein complex comprising a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity; and a guide nucleic acid comprising a sequence complementary to a portion of the target nucleic acid e) site-specifically cleaving the target nucleic acid by the chimeric polypeptide in a cancer cell in the subject; f) non-specific cleaving other nucleic acids in the cancer cell by the chimeric polypeptide, thereby inducing cell death of the cancer cell; and g) administering to the subject a terminator, thereby terminating activity of the chimeric polypeptide. 327. The method of embodiment 326, wherein the biological sample is selected from the group consisting of: blood, plasma, saliva, body fluid, tumor sample, biopsy sample, sample comprising cell-free nucleic acids, or any combination thereof 328. The method of embodiment 326, wherein the determining comprises sequencing. 329. The method of embodiment 326, wherein the target nucleic acid is absent in a healthy cell of the subject. 330. The method of embodiment 326, wherein the terminator is an antibody specific to the chimeric polypeptide. 331. The method of embodiment 326, wherein the terminator is ZnSO4. 332. The method of embodiment 326, wherein the target nucleic acid comprises RNA. 333. The method of embodiment 326, wherein the target nucleic acid comprises DNA. 334. The method of embodiment 326, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 335. The method of embodiment 334, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, and a isochromosome. 336. The method of embodiment 334, wherein the target nucleic acid comprises a translocation. 337. The method of embodiment 326, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer. 338. The method of embodiment 326, wherein the target nucleic acid comprises a sequence associated with cancer progression. 339. The method of embodiment 326, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, or HER2. 340. The method of embodiment 326, wherein the guide nucleic acid does not bind to a nucleic acid in a healthy cell. 341. The method of embodiment 326, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 342. The method of embodiment 341, wherein the non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 343. The method of embodiment 326, wherein the first domain comprises a Cas12a (or Cpf1) domain. 344. The method of embodiment 326, wherein the first domain comprises a Cas13a or Cas13b domain. 345. The method of embodiment 326, wherein the first domain comprises a Cas9 domain. 346. The method of embodiment 326, wherein the guide nucleic acid comprises a sequence selected from the group consisting of sequences in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 347. The method of embodiment 326, wherein the first domain is a CRISPR-Associated (Cas) Protein domain that comprises inducible non-specific nuclease activity. 348. The method of embodiment 347, wherein the inducible non-specific nuclease activity of the first domain is activated by site-specific cleavage of the target nucleic acid by the first domain. 349. The method of embodiment 326, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an ExoI domain, an ExoIII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 350. The method of embodiment 326, wherein the chimeric polypeptide generates a 3′ OH overhang. 351. The method of embodiment 326, wherein the chimeric polypeptide exposes a recessed 3′ OH. 352. The method of embodiment 326, wherein the second domain comprises an enzyme having cleaved end resection activity. 353. The method of embodiment 326, wherein the second domain comprises mung bean nuclease. 354. A method of selectively inducing cell death in a cell, the method comprising: a) administering a chimeric polypeptide comprising a first domain comprising sequence-specific endonuclease activity and a second domain comprising exonuclease activity a guide nucleic acid comprising a sequence complementary to a target nucleic acid in the cell; and b) cleaving the target nucleic acid, thereby inducing cell death. 355. The method of embodiments 354, wherein the target nucleic acid is associated with a disorder. 356. The method of embodiment 354, wherein the target nucleic acid is in a cancer cell of the subject. 357. The method of embodiment 354, wherein the target nucleic acid is absent in a healthy cell of the subject. 358. The method of embodiment 354, wherein the target nucleic acid comprises a sequence associated with the cancer. 359. The method of embodiment 354, wherein the target nucleic acid comprises RNA. 360. The method of embodiment 354, wherein the target nucleic acid comprises DNA. 361. The method of embodiment 354, wherein the target nucleic acid is within a region comprising a chromosomal abnormality. 362. The method of embodiment 361, wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, copy number variations, an indel, and an isochromosome. 363. The method of embodiment 354, wherein the cell is in a plurality of cells. 364. The method of embodiment 364, wherein the plurality of cells comprises a healthy cell. 365. The method of embodiment 364, wherein after the administering, the healthy cell lives. 366. The method of embodiment 364, wherein after the administering, the healthy cell proliferates. 367. The method of embodiment 354, wherein the cleaving comprises cleaving at one or more than one cleavage site in the cell. 368. The method of embodiment 355, wherein the disorder is cancer. 369. The method of embodiment 368, wherein the cancer comprises lung cancer or pancreatic cancer. 370. The method of embodiment 354, wherein the target nucleic acid comprises a single nucleotide polymorphism specific to a cancer, a translocation, or a sequence associated with cancer progression. 371. The method of embodiment 354, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of: BRCA-1, BRAF, BCR-ABL, HER2, KIF5A, IRX1, ADAMTS16, GNPDA2, KCNE2, SLC15A5, SMIM11, DACH2, HERC2P2, CD68SHBG, ERBB2, KRT16, LINC00536, TRPS1, CDK8, TRAPPC9, HERC2P2, SIRPB1, MRC1, ATP11A, POTEB, HERC2P2, PRDM9, CDKN2B, HPV, LINE2 (MT2), CCR5, or HPRT1. 372. The method of embodiment 354, wherein the chimeric polypeptide further comprises inducible non-specific nuclease activity. 373. The method of embodiment 372, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 374. The method of embodiment 354, wherein the first domain comprises a Cas12a (or Cpf1) domain, a Cas12a domain, a Cas12b domain, a Cas12c domain, a Cas12d domain, a Cas12e domain, a Cas12f domain, a Cas12g domain, a Cas12h domain, a Cas12i domain, a Cas13a domain, a Cas13b domain, a Cas14 domain, or a Cas9 domain. 375. The method of embodiment 354, wherein the first domain comprises a Cas12a (or Cpf1) domain. 376. The method of embodiment 354, wherein the first domain comprises a Cas13a or Cas13b domain. 377. The method of embodiment 354, wherein the first domain comprises a Cas9 domain. 378. The method of embodiment 354, wherein the guide nucleic acid comprises a sequence selected from the group consisting of the sequences listed in Table 6 and paragraph [00303] or Table 7 and paragraph [00305]. 379. The method of embodiment 354, wherein the second domain comprises a RecE domain, a RecJ domain, a T5 domain, a Lambda Exonuclease domain, a RecBCD domain, a DNA pol I small fragment domain, an Exo1 domain, an ExoII domain, an ExoVII domain, an Exo T domain, a Trex1 domain, or a Trex2 domain. 380. The method of embodiment 354, wherein the chimeric polypeptide generates a 3′ OH overhang. 381. The method of embodiment 354, wherein the chimeric polypeptide exposes a recessed 3′ OH. 382. The method of embodiment 354, wherein the second domain comprises an enzyme having cleaved end resection activity. 383. The method of embodiment 354, wherein the second domain comprises mung bean nuclease. 384. The method of embodiment 364, wherein the target nucleic acid is absent in a healthy cell of the subject.

EXAMPLES Example 1. Exemplary Implementation in the Context of Cancer

An individual's tumor tissues are tested to determine whether or not an appropriate target is present. Using whole genomic sequence analysis of cancer specimens from patients, SNPs or other mutations that show the sufficient specificity compared with normal cells are identified.

Multiple gRNA molecules having complementary sequence to the cancer-specific SNPs are synthesized. These synthesized gRNAs recognize SNPs specifically in cancer cell in vivo and induce the activation of genome editing and non-specific nuclease activity of CRISPR PLUS™.

Cancer cell specific activation of genome editing and non-specific nuclease function of CRISPR PLUS™ are validated using target DNA cleavage and nuclease activity assay preliminary in ex vivo systems. A selected sets of pre-assembled gRNA and CRISPR PLUS™ (including CRISPR) protein complex is administered into the patient.

Through the administration of the selected sets of pre-assembled gRNA and CRISPR PLUS™, the CRISPR PLUS™-gRNA complexes enter the cells and screen the genomic DNA to detect complementary SNP sequences in the cells. In normal cells containing no SNP or other mutant sequence, the CRISPR PLUS™-gRNA complexes are not activated, and as a result, little or no targeted or untargeted cell nucleic acid degradation occurs. However, in cancer cells containing SNPs or other mutant sequences that are complementary to the gRNAs, the genome editing function is activated, and subsequently non-specific nuclease activity of the CRISPR PLUS™-gRNA complex is activated.

The activated genome editing function removes the nucleic acids harboring the cancer specific SNP or other mutation or mutant sequence. The activated non-specific nuclease function then degrades cellular double stranded and single stranded DNA/RNA molecules which in turn induce cell death in cancer specific manner (see FIG. 2).

The administration regimen may be followed by administration of a ribonucleoprotein complex such as a CRISPR or CRISPR PLUS™ terminator to remove the activity of CRISPR PLUS™-gRNA complex remaining in a subject's body. Then the anti-Cas antibody is subsequently administered to neutralize the effects of the CRISPR or CRISPR PLUS™ proteins. This termination process of CRISPR or CRISPR PLUS™-gRNA complex after completion of therapy minimizes any potential side effects caused by off-target activity of CRISPR or CRISPR PLUS™.

Example 2. Representative Cas9 Nucleic Acid and Protein

A number of CRISPR/Cas complexes are consistent with the disclosure herein. An exemplary Cas sequence, that of SpyCas9, is provided below in nucleotide and amino acid form.

>NC_002737.2:854751-858857 Streptococcus pyogenes M1 GAS (SEQ ID NO: 67) ATGGATAAGAAATACTCAATAGGCTTAGATATCGG CACAAATAGCGTCGGATGGGCGGTGATCACTGATG AATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTG GGAAATACAGACCGCCACAGTATCAAAAAAAATCT TATAGGGGCTCTTTTATTTGACAGTGGAGAGACAG CGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGA AGGTATACACGTCGGAAGAATCGTATTTGTTATCT ACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAG ATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTT TTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC TATTTTTGGAAATATAGTAGATGAAGTTGCTTATC ATGAGAAATATCCAACTATCTATCATCTGCGAAAA AAATTGGTAGATTCTACTGATAAAGCGGATTTGCG CTTAATCTATTTGGCCTTAGCGCATATGATTAAGT TTCGTGGTCATTTTTTGATTGAGGGAGATTTAAAT CCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAA ACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCG ATTCTTTCTGCACGATTGAGTAAATCAAGACGATT AGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA AAAATGGCTTATTTGGGAATCTCATTGCTTTGTCA TTGGGTTTGACCCCTAATTTTAAATCAAATTTTGA TTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAG ATACTTACGATGATGATTTAGATAATTTATTGGCG CAAATTGGAGATCAATATGCTGATTTGTTTTTGGC AGCTAAGAATTTATCAGATGCTATTTTACTTTCAG ATATCCTAAGAGTAAATACTGAAATAACTAAGGCT CCCCTATCAGCTTCAATGATTAAACGCTACGATGA ACATCATCAAGACTTGACTCTTTTAAAAGCTTTAG TTCGACAACAACTTCCAGAAAAGTATAAAGAAATC TTTTTTGATCAATCAAAAAACGGATATGCAGGTTA TATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGT ACTGAGGAATTATTGGTGAAACTAAATCGTGAAGA TTTGCTGCGCAAGCAACGGACCTTTGACAACGGCT CTATTCCCCATCAAATTCACTTGGGTGAGCTGCAT GCTATTTTGAGAAGACAAGAAGACTTTTATCCATT TTTAAAAGACAATCGTGAGAAGATTGAAAAAATCT TGACTTTTCGAATTCCTTATTATGTTGGTCCATTG GCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG GAAGTCTGAAGAAACAATTACCCCATGGAATTTTG AAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCA TTTATTGAACGCATGACAAACTTTGATAAAAATCT TCCAAATGAAAAAGTACTACCAAAACATAGTTTGC TTTATGAGTATTTTACGGTTTATAACGAATTGACA AAGGTCAAATATGTTACTGAAGGAATGCGAAAACC AGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTG TTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAGAAGATTATTTCAAAAAAAT AGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG AAGATAGATTTAATGCTTCATTAGGTACCTACCAT GATTTGCTAAAAATTATTAAAGATAAAGATTTTTT GGATAATGAAGAAAATGAAGATATCTTAGAGGATA TTGTTTTAACATTGACCTTATTTGAAGATAGGGAG ATGATTGAGGAAAGACTTAAAACATATGCTCACCT CTTTGATGATAAGGTGATGAAACAGCTTAAACGTC GCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAA TTGATTAATGGTATTAGGGATAAGCAATCTGGCAA AACAATATTAGATTTTTTGAAATCAGATGGTTTTG CCAATCGCAATTTTATGCAGCTGATCCATGATGAT AGTTTGACATTTAAAGAAGACATTCAAAAAGCACA AGTGTCTGGACAAGGCGATAGTTTACATGAACATA TTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAA GGTATTTTACAGACTGTAAAAGTTGTTGATGAATT GGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATA TCGTTATTGAAATGGCACGTGAAAATCAGACAACT CAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAA ACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTC AGATTCTTAAAGAGCATCCTGTTGAAAATACTCAA TTGCAAAATGAAAAGCTCTATCTCTATTATCTCCA AAATGGAAGAGACATGTATGTGGACCAAGAATTAG ATATTAATCGTTTAAGTGATTATGATGTCGATCAC ATTGTTCCACAAAGTTTCCTTAAAGACGATTCAAT AGACAATAAGGTCTTAACGCGTTCTGATAAAAATC GTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAAGATGAAAAACTATTGGAGACAACTTCT AAACGCCAAGTTAATCACTCAACGTAAGTTTGATA ATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAA CTTGATAAAGCTGGTTTTATCAAACGCCAATTGGT TGAAACTCGCCAAATCACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAA AATGATAAACTTATTCGAGAGGTTAAAGTGATTAC CTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAG ATTTCCAATTCTATAAAGTACGTGAGATTAACAAT TACCATCATGCCCATGATGCGTATCTAAATGCCGT CGTTGGAACTGCTTTGATTAAGAAATATCCAAAAC TTGAATCGGAGTTTGTCTATGGTGATTATAAAGTT TATGATGTTCGTAAAATGATTGCTAAGTCTGAGCA AGAAATAGGCAAAGCAACCGCAAAATATTTCTTTT ACTCTAATATCATGAACTTCTTCAAAACAGAAATT ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCT AATCGAAACTAATGGGGAAACTGGAGAAATTGTCT GGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAA GTATTGTCCATGCCCCAAGTCAATATTGTCAAGAA AACAGAAGTACAGACAGGCGGATTCTCCAAGGAGT CAATTTTACCAAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGG TGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCC TAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAG AAGTTAAAATCCGTTAAAGAGTTACTAGGGATCAC AATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGA TTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTT AAAAAAGACTTAATCATTAAACTACCTAAATATAG TCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGC TGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAG CTGGCTCTGCCAAGCAAATATGTGAATTTTTTATA TTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTC CAGAAGATAACGAACAAAAACAATTGTTTGTGGAG CAGCATAAGCATTATTTAGATGAGATTATTGAGCA AATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATATAAC AAACATAGAGACAAACCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTG GAGCTCCCGCTGCTTTTAAATATTTTGATACAACA ATTGATCGTAAACGATATACGTCTACAAAAGAAGT TTTAGATGCCACTCTTATCCATCAATCCATCACTG GTCTTTATGAAACACGCATTGATTTGAGTCAGCTA GGAGGTGACTGA >SpyCas9 protein (SEQ ID NO: 68) MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK KAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSD FRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFE KNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR KRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

Example 3. Representative Alternative to Cas9—FnCpf1 Nucleic Acid and Protein

An exemplary Cas alternative, Cpf1, is provided below in nucleotide and amino acid form.

>FnCpf1, NCBIaccessionno.CP009633.1 (SEQ ID NO: 69) ATGTCAATTTATCAAGAATTTGTTAATAAATATAG TTTAAGTAAAACTCTAAGATTTGAGTTAATCCCAC AGGGTAAAACACTTGAAAACATAAAAGCAAGAGGT TTGATTTTAGATGATGAGAAAAGAGCTAAAGACTA CAAAAAGGCTAAACAAATAATTGATAAATATCATC AGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGT ATTAGCGAAGATTTATTACAAAACTATTCTGATGT TTATTTTAAACTTAAAAAGAGTGATGATGATAATC TACAAAAAGATTTTAAAAGTGCAAAAGATACGATA AAGAAACAAATATCTGAATATATAAAGGACTCAGA GAAATTTAAGAATTTGTTTAATCAAAACCTTATCG ATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTA TGGCTAAAGCAATCTAAGGATAATGGTATAGAACT ATTTAAAGCCAATAGTGATATCACAGATATAGATG AGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGG ACAACTTATTTTAAGGGTTTTCATGAAAATAGAAA AAATGTTTATAGTAGCAATGATATTCCTACATCTA TTATTTATAGGATAGTAGATGATAATTTGCCTAAA TTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAA AGACAAAGCTCCAGAAGCTATAAACTATGAACAAA TTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGAT ATTGACTACAAAACATCTGAAGTTAATCAAAGAGT TTTTTCACTTGATGAAGTTTTTGAGATAGCAAACT TTAATAATTATCTAAATCAAAGTGGTATTACTAAA TTTAATACTATTATTGGTGGTAAATTTGTAAATGG TGAAAATACAAAGAGAAAAGGTATAAATGAATATA TAAATCTATACTCACAGCAAATAAATGATAAAACA CTCAAAAAATATAAAATGAGTGTTTTATTTAAGCA AATTTTAAGTGATACAGAATCTAAATCTTTTGTAA TTGATAAGTTAGAAGATGATAGTGATGTAGTTACA ACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTT TAAAACAGTAGAAGAAAAATCTATTAAAGAAACAC TATCTTTATTATTTGATGATTTAAAAGCTCAAAAA CTTGATTTGAGTAAAATTTATTTTAAAAATGATAA ATCTCTTACTGATCTATCACAACAAGTTTTTGATG ATTATAGTGTTATTGGTACAGCGGTACTAGAATAT ATAACTCAACAAATAGCACCTAAAAATCTTGATAA CCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAA AAACTGAAAAAGCAAAATACTTATCTCTAGAAACT ATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAG AGATATAGATAAACAGTGTAGGTTTGAAGAAATAC TTGCAAACTTTGCGGCTATTCCGATGATATTTGAT GAAATAGCTCAAAACAAAGACAATTTGGCACAGAT ATCTATCAAATATCAAAATCAAGGTAAAAAAGACC TACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCT ATCAAGGATCTTTTAGATCAAACTAATAATCTCTT ACATAAACTAAAAATATTTCATATTAGTCAGTCAG AAGATAAGGCAAATATTTTAGACAAGGATGAGCAT TTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCT AGCGAATATAGTGCCTCTTTATAACAAAATTAGAA ACTATATAACTCAAAAGCCATATAGTGATGAGAAA TTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAA TGGTTGGGATAAAAATAAAGAGCCTGACAATACGG CAATTTTATTTATCAAAGATGATAAATATTATCTG GGTGTGATGAATAAGAAAAATAACAAAATATTTGA TGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTT ATAAAAAAATTGTTTATAAACTTTTACCTGGCGCA AATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAA ATCTATAAAATTTTATAATCCTAGTGAAGATATAC TTAGAATAAGAAATCATTCCACACATACAAAAAAT GGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTT TAATATTGAAGATTGCCGAAAATTTATAGATTTTT ATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAA GATTTTGGATTTAGATTTTCTGATACTCAAAGATA TAATTCTATAGATGAATTTTATAGAGAAGTTGAAA ATCAAGGCTACAAACTAACTTTTGAAAATATATCA GAGAGCTATATTGATAGCGTAGTTAATCAGGGTAA ATTGTACCTATTCCAAATCTATAATAAAGATTTTT CAGCTTATAGCAAAGGGCGACCAAATCTACATACT TTATATTGGAAAGCGCTGTTTGATGAGAGAAATCT TCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAG AGCTTTTTTATCGTAAACAATCAATACCTAAAAAA ATCACTCACCCAGCTAAAGAGGCAATAGCTAATAA AAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTG AATATGATTTAATCAAAGATAAACGCTTTACTGAA GATAAGTTTTTCTTTCACTGTCCTATTACAATCAA TTTTAAATCTAGTGGAGCTAATAAGTTTAATGATG AAATCAATTTATTGCTAAAAGAAAAAGCAAATGAT GTTCATATATTAAGTATAGATAGAGGTGAAAGACA TTTAGCTTACTATACTTTGGTAGATGGTAAAGGCA ATATCATCAAACAAGATACTTTCAACATCATTGGT AATGATAGAATGAAAACAAACTACCATGATAAGCT TGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGA AAGACTGGAAAAAGATAAATAACATCAAAGAGATG AAAGAGGGCTATCTATCTCAGGTAGTTCATGAAAT AGCTAAGCTAGTTATAGAGTATAATGCTATTGTGG TTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGG CGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTT AGAAAAAATGCTAATTGAGAAACTAAACTATCTAG TTTTCAAAGATAATGAGTTTGATAAAACTGGGGGA GTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGA GACTTTTAAAAAGATGGGTAAACAAACAGGTATTA TCTACTATGTACCAGCTGGTTTTACTTCAAAAATT TGTCCTGTAACTGGTTTTGTAAATCAGTTATATCC TAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCT TTAGTAAGTTTGACAAGATTTGTTATAACCTTGAT AAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAA CTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTA TAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGA AATTCAGATAAAAATCATAATTGGGATACTCGAGA AGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAA AAGATTATTCTATCGAATATGGGCATGGCGAATGT ATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAA GTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTA TCTTACAAATGCGTAACTCAAAAACAGGTACTGAG TTAGATTATCTAATTTCACCAGTAGCAGATGTAAA TGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAA ATATGCCTCAAGATGCTGATGCCAATGGTGCTTAT CATATTGGGCTAAAAGGTCTGATGCTACTAGGTAG GATCAAAAATAATCAAGAGGGCAAAAAACTCAATT TGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTG CAGAATAGGAATAACTAA >FnCpf1, NCBIaccessionno.CP009633.1 protein sequence (SEQ ID NO: 70) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND VHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIG NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM KEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRG RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE LDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV QNRNN

Example 4. Exonucleases Used in Various Ribonucleoprotein Nuclease Complexes

A number of exonucleases are consistent with the disclosure herein. A partial listing of exonucleases is provided below, although others are also consistent with the disclosure herein.

>Exoribonuclease T (SEQ ID NO: 71) MSDNAQLTGLCDRFRGFYPVVIDVETAGFNAKTDA LLEIAAITLKMDEQGWLMPDTTLHFHVEPFVGANL QPEALAFNGIDPNDPDRGAVSEYEALHEIFKVVRK GIKASGCNRAIMVAHNANFDHSFMMAAAERASLKR NPFHPFATFDTAALAGLALGQTVLSKACQTAGMDF DSTQAHSALYDTERTAVLFCEIVNRWKRLGGWPLS AAEEV >TREX2 (SEQ ID NO: 72) MSEAPRAETFVFLDLEATGLPSVEPEIAELSLFAV HRSSLENPEHDESGALVLPRVLDKLTLCMCPERPF TAKASEITGLSSEGLARCRKAGFDGAVVRTLQAFL SRQAGPICLVAHNGFDYDFPLLCAELRRLGARLPR DTVCLDTLPALRGLDRAHSHGTRARGRQGYSLGSL FHRYFRAEPSAAHSAEGDVHTLLLIFLHRAAELLA WADEQARGWAHIEPMYLPPDDPSLEA >TREX1 (SEQ ID NO: 73) MQTLIFFDMEATGLPFSQPKVTELCLLAVHRCALE SPPTSQGPPPTVPPPPRVVDKLSLCVAPGKACSPA ASEITGLSTAVLAAHGRQCFDDNLANLLLAFLRRQ PQPWCLVAHNGDRYDFPLLQAELAMLGLTSALDGA FCVDSITALKALERASSPSEHGPRKSYSLGSIYTR LYGQSPPDSHTAEGDVLALLSICQWRPQALLRWVD AHARPFGTIRPMYGVTASARTKPRPSAVTTTAHLA TTRNTSPSLRESRGTKDLPPVKDPGALSREGLLAP LGLLAILTLAVATLYGLSLATPGD >RecBCD_RecB (SEQ ID NO: 74) MSDVAETLDPLRLPLQGERLIEASAGTGKTFTIAA LYLRLLLGLGGSAAFPRPLTVEELLVVTFTEAATA ELRGRIRSNIHELRIACLRETTDNPLYERLLEEID DKAQAAQWLLLAERQMDEAAVFTIHGFCQRMLNLN AFESGMLFEQQLIEDESLLRYQACADFWRRHCYPL PREIAQVVFETWKGPQALLRDINRYLQGEAPVIKA PPPDDETLASRHAQIVARIDTVKQQWRDAVGELDA LIESSGIDRRKFNRSNQAKWIDKISAWAEEETNSY QLPESLEKFSQRFLEDRTKAGGETPRHPLFEAIDQ LLAEPLSIRDLVITRALAEIRETVAREKRRRGELG FDDMLSRLDSALRSESGEVLAAAIRTRFPVAMIDE FQDTDPQQYRIFRRIWHHQPETALLLIGDPKQAIY AFRGADIFTYMKARSEVHAHYTLDTNWRSAPGMVN SVNKLFSQTDDAFMFREIPFIPVKSAGKNQALRFV FKGETQPAMKMWLMEGESCGVGDYQSTMAQVCAAQ IRDWLQAGQRGEALLMNGDDARPVRASDISVLVRS RQEAAQVRDALTLLEIPSVYLSNRDSVFETLEAQE MLWLLQAVMTPERENTLRSALATSMMGLNALDIET LNNDEHAWDVVVEEFDGYRQIWRKRGVMPMLRALM SARNIAENLLATAGGERRLTDILHISELLQEAGTQ LESEHALVRWLSQHILEPDSNASSQQMRLESDKHL VQIVTIHKSKGLEYPLVWLPFITNFRVQEQAFYHD RHSFEAVLDLNAAPESVDLAEAERLAEDLRLLYVA LTRSVWHCSLGVAPLVRRRGDKKGDTDVHQSALGR LLQKGEPQDAAGLRTCIEALCDDDIAWQTAQTGDN QPWQVNDVSTAELNAKTLQRLPGDNWRVISYSGLQ QRGHGIAQDLMPRLDVDAAGVASVVEEPTLTPHQF PRGASPGTFLHSLFEDLDFTQPVDPNWVREKLELG GFESQWEPVLTEWITAVLQAPLNETGVSLSQLSAR NKQVEMEFYLPISEPLIASQLDTLIRQFDPLSAGC PPLEFMQVRGMLKGFIDLVFRHEGRYYLLDYKSNW LGEDSSAYTQQAMAAAMQAHRYDLQYQLYTLALHR YLRHRIADYDYEHHFGGVIYLFLRGVDKEHPQQGI YTTRPNAGLIALMDEMFAGMTLEEA >RecBCD_RecC (SEQ ID NO: 75) MLRVYHSNRLDVLEALMEFIVERERLDDPFEPEMI LVQSTGMAQWLQMTLSQKFGIAANIDFPLPASFIW DMFVRVLPEIPKESAFNKQSMSWKLMTLLPQLLER EDFTLLRHYLTDDSDKRKLFQLSSKAADLFDQYLV YRPDWLAQWETGHLVEGLGEAQAWQAPLWKALVEY THQLGQPRWHRANLYQRFIETLESATTCPPGLPSR VFICGISALPPVYLQALQALGKHIEIHLLFTNPCR YYWGDIKDPAYLAKLLTRQRRHSFEDRELPLFRDS ENAGQLFNSDGEQDVGNPLLASWGKLGRDYIYLLS DLESSQELDAFVDVTPDNLLHNIQSDILELENRAV AGVNIEEFSRSDNKRPLDPLDSSITFHVCHSPQRE VEVLHDRLLAMLEEDPILTPRDIIVMVADIDSYSP FIQAVFGSAPADRYLPYAISDRRARQSHPVLEAFI SLLSLPDSRFVSEDVLALLDVPVLAARFDITEEGL RYLRQWVNESGIRWGIDDDNVRELELPATGQHTWR FGLTRMLLGYAMESAQGEWQSVLPYDESSGLIAEL VGHLASLLMQLNIWRRGLAQERPLEEWLPVCRDML NAFFLPDAETEAAMTLIEQQWQAIIAEGLGAQYGD AVPLSLLRDELAQRLDQERISQRFLAGPVNICTLM PMRSIPFKVVCLLGMNDGVYPRQLAPLGFDLMSQK PKRGDRSRRDDDRYLFLEALISAQQKLYISYIGRS IQDNSERFPSVLVQELIDYIGQSHYLPGDEALNCD ESEARVKAHLTCLHTRMPFDPQNYQPGERQSYARE WLPAASQAGKAHSEFVQPLPFTLPETVPLETLQRF WAHPVRAFFQMRLQVNFRTEDSEIPDTEPFILEGL SRYQINQQLLNALVEQDDAERLFRRFRAAGDLPYG AFGEIFWETQCQEMQQLADRVIACRQPGQSMEIDL ACNGVQITGWLPQVQPDGLLRWRPSLLSVAQGMQL WLEHLVYCASGGNGESRLFLRKDGEWRFPPLAAEQ ALHYLSQLIEGYREGMSAPLLVLPESGGAWLKTCY DAQNDAMLDDDSTLQKARTKFLQAYEGNMMVRGEG DDIWYQRLWRQLTPETMEAIVEQSQRFLLPLFRFN QS >RecBCD_RecD (SEQ ID NO: 76) MKLQKQLLEAVEHKQLRPLDVQFALTVAGDEHPAV TLAAALLSHDAGEGHVCLPLSRLENNEASHPLLAT CVSEIGELQNWEECLLASQAVSRGDEPTPMILCGD RLYLNRMWCNERTVARFFNEVNHAIEVDEALLAQT LDKLFPVSDEINWQKVAAAVALTRRISVISGGPGT GKTTTVAKLLAALIQMADGERCRIRLAAPTGKAAA RLTESLGKALRQLPLTDEQKKRIPEDASTLHRLLG AQPGSQRLRHHAGNPLHLDVLVVDEASMIDLPMMS RLIDALPDHARVIFLGDRDQLASVEAGAVLGDICA YANAGFTAERARQLSRLTGTHVPAGTGTEAASLRD SLCLLQKSYRFGSDSGIGQLAAAINRGDKTAVKTV FQQDFTDIEKRLLQSGEDYIAMLEEALAGYGRYLD LLQARAEPDLIIQAFNEYQLLCALREGPFGVAGLN ERIEQFMQQKRKIHRHPHSRWYEGRPVMTARNDSA LGLFNGDIGIALDRGQGTRVWFAMPDGNIKSVQPS RLPEHETTWAMTVHKSQGSEFDHAALILPSQRTPV VTRELVYTAVTRARRRLSLYADERILSAAIATRTE RRSGLAALFSSRE >Exodeoxyribonuclease I (SEQ ID NO: 77) MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIR TDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGIT PQEARAKGENEAAFAARIHSLFTVPKTCILGYNNV RFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDV MRACYALRPEGINWPENDDGLPSFRLEHLTKANGI EHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFT HRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSW VAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLR ERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTL RPEDADRLGINRQHCLDNLKILRENPQVREKVVAI FAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLE TEPRNLPALDITFVDKRIEKLLFNYRARNFPGILD YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYA DDKEKVALLKALWQYAEEIV >Exodeoxyribonuclease III (SEQ ID NO: 78) MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQET KVHDDMFPLEEVAKLGYNVFYHGQKGHYGVALLTK ETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVIV INGYFPQGESRDHPIKFPAKAQFYQNLQNYLETEL KRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTG KCSFLPEEREWMDRLMSWGLVDTFRHANPQTADRF SWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETG IDYEIRSMEKPSDHAPVWATFRR >Mungbean exonuclease (SEQ ID NO: 79) MQTLQMSLLTQPYVQPRFPCKRYPTFSASCRTQKT AITKTEKVFFSESFDQTRCTQPLSEKKKRVFFLDV NPLCYEGSKPSLRSFGRWLSLFLHQVSLTDPVIAV IDGEGGSEHRRKLLPSYKAHRKKFMRHMSSGHVGR SHQVINDVLGKCNVPVIKVAGHEADDVVATLAGQV VNKGFRVVIGSPDKDFKQLISEDVQIVMPLPELQR WSFYTLRHYRDQYNCDPESDLSFRCIVGDEVDGVP GIQHLVPSFGRKTAMKLIKKHGSLETLLNAAAIRT VGRPYAQDALKNHADYLRRNYEVLALKRDVNIQLY DEWLVKRDNHNDKTALSSFFKYLGESKELSYNGRP ISYNG >RecJ (SEQ ID NO: 80) VKQQIQLRRREVDETADLPAELPPLLRRLYASRGV RSAQELERSVKGMLPWQQLSGVEKAVEILYNAFRE GTRIIVVGDFDADGATSTALSVLAMRSLGCSNIDY LVPNRFEDGYGLSPEVVDQAHARGAQLIVTVDNGI SSHAGVEHARSLGIPVIVTDHHLPGDTLPAAEAII NPNLRDCNFPSKSLAGVGVAFYLMLALRTFLRDQG WFDERNIAIPNLAELLDLVALGTVADVVPLDANNR ILTWQGMSRIRAGKCRPGIKALLEVANRDAQKLAA SDLGFALGPRLNAAGRLDDMSVGVALLLCDNIGEA RVLANELDALNQTRKEIEQGMQIEALTLCEKLERS RDTLPGGLAMYHPEWHQGVVGILASRIKERFHRPV IAFAPAGDGTLKGSGRSIQGLHMRDALERLDTLYP GMMLKFGGHAMAAGLSLEEDKFKLFQQRFGELVTE WLDPSLLQGEVVSDGPLSPAEMTMEVAQLLRDAGP WGQMFPEPLFDGHFRLLQQRLVGERHLKVMVEPVG GGPLLDGIAFNVDTALWPDNGVREVQLAYKLDINE FRGNRSLQIIIDNIWPI >RecE (SEQ ID NO: 81) MSTKPLFLLRKAKKSSGEPDVVLWASNDFESTCAT LDYLIVKSGKKLSSYFKAVATNFPVVNDLPAEGEI DFTWSERYQLSKDSMTWELKPGAAPDNAHYQGNTN VNGEDMTEIEENMLLPISGQELPIRWLAQHGSEKP VTHVSRDGLQALHIARAEELPAVTALAVSHKTSLL DPLEIRELHKLVRDTDKVFPNPGNSNLGLITAFFE AYLNADYTDRGLLTKEWMKGNRVSHITRTASGANA GGGNLTDRGEGFVHDLTSLARDVATGVLARSMDLD IYNLHPAHAKRIEEIIAENKPPFSVFRDKFITMPG GLDYSRAIVVASVKEAPIGIEVIPAHVTEYLNKVL TETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQD DEEKPQPSGTTAVEQGEAETMEPDATEHHQDTQPL DAQSQVNSVDAKYQELRAELHEARKNIPSKNPVDD DKLLAASRGEFVDGISDPNDPKWVKGIQTRDCVYQ NQPETEKTSPDMNQPEPVVQQEPEIACNACGQTGG DNCPDCGAVMGDATYQETFDEESQVEAKENDPEEM EGAEHPHNENAGSDPHRDCSDETGEVADPVIVEDI EPGIYYGISNENYHAGPGISKSQLDDIADTPALYL WRKNAPVDTTKTKILDLGTAFHCRVLEPEEFSNRF IVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAE EGRKIELMYQSVMALPLGQWLVESAGHAESSIYWE DPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFK TAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVAS TTIECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLS DCLNTDEWPAIKTLSLPRWAKEYAND >T5 (SEQ ID NO: 82) MASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVST IQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGN RDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFP TFTIRGVEADDMAAYIVKLIGHLYDHVWLISTDGD WDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVE QFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFG NVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILV DLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ >Lambda exonuclease (SEQ ID NO: 83) MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITAS EVHNVIAKPRSGKKWPDMKMSYFHTLLAEVCTGVA PEVNAKALAWGKQYENDARTLFEFTSGVNVTESPI IYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFM KFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANY DPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKM DEALAEIGFVFGEQWR >Exonuclease VII small unit (SEQ ID NO: 84) MPKKNEAPASFEKALSELEQIVTRLESGDLPLEEA LNEFERGVQLARQGQAKLQQAEQRVQILLSDNEDA SLTPFTPDNE >Exonuclease VII large unit (SEQ ID NO: 85) MLPSQSPAIFTVSRLNQTVRLLLEHEMGQVWISGE ISNFTQPASGHWYFTLKDDTAQVRCAMFRNSNRRV TFRPQHGQQVLVRANITLYEPRGDYQIIVESMQPA GEGLLQQKYEQLKAKLQAEGLFDQQYKKPLPSPAH CVGVITSKTGAALHDILHVLKRRDPSLPVIIYPAA VQGDDAPGQIVRAIELANQRNECDVLIVGRGGGSL EDLWSFNDERVARAIFTSRIPVVSAVGHETDVTIA DFVADLRAPTPSAAAEVVSRNQQELLRQVQSTRQR LEMAMDYYLANRTRRFTQIHHRLQQQHPQLRLARQ QTMLERLQKRMSFALENQLKRTGQQQQRLTQRLNQ QNPQPKIHRAQTRIQQLEYRLAETLRAQLSATRER FGNAVTHLEAVSPLSTLARGYSVTTATDGNVLKKV KQVKAGEMLTTRLEDGWIESEVKNIQPVKKSRKKV H

Example 5. In Vitro Ribonucleoprotein Specificity Assays

CRISPR/Cas12a nuclease complexes were assembled around a DHCR7 nucleic acid guide. Activity was assayed against DNA #1, comprising target sequence, and DNA #2, a non-target control. Output is measured by gel electrophoresis to assay for endonucleolytic activity, evidenced by presence of the ‘cleaved DNA #1’ band, and exonuclease activity, evidenced by band absence. Activity is assayed at 1.5 hours (top) and 24 hours (bottom) of FIG. 3.

Viewing the results from the left, one sees a DNA ladder followed by a nucleic acid control having only DNA #1 and DNA #2. At lane 3 one sees an assembled ribonucleoprotein complex with substrate indicating activity. At 1.5 hours endonucleolytic activity is apparent by the presence of the DNA #1 cleaved band, while at 24 hours a combination of endonucleolytic and exonucleolytic activity has eliminated both the cleaved and uncleaved DNA #1 bands. Lane 4 indicates that no off-target endonuclease or exonuclease activity occurs in the absence of an assembled complex having a guide RNA that has been activated by an active target sequence molecule, in this case DNA #1. Lane 5 indicates that no off-target endonuclease or exonuclease activity occurs in the presence of unassembled complex. At lane 6, one sees that activated complexes exhibit nonspecific nuclease activity at 24 hours but not at 1.5 hours. Lane 7 indicates that complexes lacking a guide RNA do not exhibit exonuclease activity.

These results indicate the benefit of having both sequence specificity and temporal specificity in CRISPR/Cas ribonucleoprotein complexes to effect specific degradation of target nucleic acids, such as in cancer or pathogen cells.

Example 6. In Vivo Ribonucleoprotein Specificity Assays

To investigate whether CRISPR/Cas nucleases have cytotoxic effect to the cells in their specific nuclease activity-dependent manner, the viability of the human immortalized cells that were transfected with CRISPR/Cas12a (Cpf1) nuclease were assessed in a time-dependent manner. To induce specific targeting activity of the nuclease, two crRNAs were used, a highly specific one (DHCR7) that specifically targets human DHCR7 gene, and a second guide (OsDwarf5_1) that is specific to rice dwarf5 gene, and that partially binds to sequence in human genome.

HeLa and HEK293 cells were transfected with CRISPR/Cas12a, two crRNAs (DHCR7 and OsDwarf5_1) or their RNP complexes. In 24, 48 and 72 hours after transfection, cell viability was assessed by cell viability assay using WST-1 reagent. Results are presented in FIG. 4.

The results showed that the viability of the cells transfected with CRISPR/Cas12a or crRNAs alone was not changed in all 24, 48 and 72 hours after transfection, suggesting that the nuclease or crRNAs alone does not have toxic effect to cells. In contrast, the viability of cells transfected with RNP complexes that have crRNA-guided DNA targeting activity was sharply decreased in 72 hours after transfection, which suggest that CRISPR/Cas12a is cytotoxic to cells depending on its specific DNA targeting nuclease activity. Both specific and less specific crRNAs have similar toxic effect to cells.

The viability of the similarly transfected cells was not changed (HEK293) or slightly decreased (HeLa) in 24 and 48 hours after transfection, suggesting that the toxicity need a certain period of time to affect the cells.

Considering that the double strand DNA breakage of target sequence by CRISPR/Cas nuclease in the cell increased from 24 to 48 hours after transfection, which subsequently induce the cytotoxic nonspecific nuclease activity, the toxicity shown in 72 hours after transfection suggest that it is caused by functions associated with specific nuclease activities of CRISPR/Cas12a rather than by a nuclease-independent indirect effect.

Taken together, the results suggest that CRISPR/Cas12a nuclease possesses cytotoxic effect to the cell, which depends on crRNA-mediated specific sequence targeting activity.

The results also re-emphasize that temporal restraints on CRISPR/Cas complexes increase specificity by decreasing off-target toxicity that is observed at 72 hours.

Experimental conditions for the assays were as follows. Human cell transfection HeLa and HEK293 cells were cultured in Dulbeco's Modified Eagle's Media (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) at 37° C., 5% CO₂ incubator. 2.5X104 cells were plated in each well of 96 well plate. 1.2 nM (100 ng) or 2.4 nM (200 ng) CRISPR/Cas nuclease and 1.2 nM (25 ng) or 2.4 nM (50 ng) crRNAs were used to form the RNP complex. Each combination of nuclease, crRNA and RNP complex were transfected to cells using Lipofectamine CRISPRMAX reagent (Thermo Fisher Scientific INC.) following the manufacturer's instruction. Briefly, in one tube, each combination of nuclease, crRNA and RNP complexes were incubated with 1.7 μl of Lipofectamine Cas Plus reagent in 5 μl of Opti-MEM at room temperature (RT) for 5 minutes. In another tube, 0.3 μl of Lipofectamine CRISPRMAX reagent was mixed with 5 μl of Opti-MEM and incubated at RT for 5 minutes. Two solutions were mixed, and incubated at RT for another 10 minutes. Then the mixed solution was directly added to cells in one well of 96 well plate. In 24, 48 and 72 hours after transfection, the cell viability was assessed using WST-1 reagent.

Cell viability assay 10 μl of WST-1 reagent (Roche #0501594401) was added to each well of 96 well plate. The plate was incubated in 37 C, 5% CO₂ incubator until the light red color of the media changed to dark red. The media volume of each well was 100 μl. The absorbance of the samples against a background control as blank was measured using FLUOstar OMEGA ELISA reader (BMG Labtech) at wavelengths of 420˜480 nm.

Example 7. In Vivo Cell Targeting

An individual is identified as suffering from cancer. The cancer may be localized to a tumor. The tumor is sequenced and a number of mutations are identified as corresponding with at least some of the tumor cells.

Further, a mutation present in a majority of the tumor cells is identified and used to specify a target sequence. A guide RNA is then synthesized to identify the target sequence and is assembled into a ribonucleoprotein complex. The complex is then administered to the patient and symptom amelioration and tumor size reduction is observed.

Example 8. Follow-on Treatment

The individual of Example 7 may continue to exhibit some symptoms, including persistent tumor growth. In such a case, the treated, reduced tumor may be resequenced. It is observed that the target sequence is no longer present, but that additional mutations may be identified.

As such, a mutation present in a majority of the tumor cells is identified and used to specify a target sequence. A guide RNA is then synthesized to identify the target sequence and is assembled into a ribonucleoprotein complex. The complex is then administered to the patient and symptom amelioration and tumor size reduction is observed.

Example 9. Follow-on Treatment

As another example, a patient suffers from leukemia. The patient is administered with the treatment imatinib mesylate (Gleevec) Some symptom amelioration is observed, but disease progression continues and the patient suffers some side effects of the treatment.

In such a case, cancer cells are sequenced. It may be observed that a genomic rearrangement resulting in coding capacity for the BCR-ABL fusion protein has occurred.

A guide RNA is then designed to target the BCR-ABL junction. That is, the guide RNA is synthesized to identify the target sequence and is assembled into a ribonucleoprotein complex. The assembled complex is then administered to the patient and symptom amelioration and leukemia remission is observed.

Example 10. Guide Nucleic Acid Design

This example describes an illustrative method for designing a guide nucleic acid for targeting a nucleic acid associated with a disorder such as cancer.

Data sources used for this example include:

-   -   1. Human reference genome sequences (e.g., hg19, GRCh37)     -   2. Mutation information of Cancer cell lines from Cancer Cell         Line Encyclopedia (CCLE)     -   3. 410 Cancer mutations listed in MSK-IMPACT™ (Memorial Sloan         Kettering Cancer Center—integrated mutation profiling of         actionable cancer targets)

Input value included CCLE mutation information, target gene ID, target gene sequence, gRNA handle sequence, crRNA length (N), PAM (Protospacer adjacent motif) sequence, and PAM location (up- or downstream).

Guide Nucleic Acid Design—Mutation locations in CCLE (Cancer Cell Line Encyclopedia) model cell line were searched in human reference genome sequences. Sequence information of nucleic acid regions that were about 30 base pair upstream and downstream of the mutation location were determined. A PAM sequence was searched in the targeted region. The location of the PAM sequence and crRNA sequence was determined. The crRNA sequence included the mutation location. The distance between the PAM sequence of the crRNA and the mutation location was calculated. A guide RNA sequence was designed by combining the crRNA sequence and the guide RNA handle sequence.

Output information associated with the designed guide RNA obtained from this method included the following:

-   1. Gene ID: NCBI GenBank Identifier -   2. Gene symbol: Gene nomenclature from HUGO European Bioinformatics     Institute (EMBL-EBI) -   3. Human genome assembly version (GRCh): Version of human genome     assembly from Genome Reference Consortium Human Build -   4. Chromosome number: The number of chromosome pair where target     gene locate -   5. Mutation start position: Start position of mutation in human     reference genome -   6. Mutation end position: End position of mutation in human     reference genome -   7. Mutation class: Class of mutation -   8. Mutation type: Type of mutation, for example, insertion or     deletion or single nucleotide polymorphism (SNP) or dinucleotide     polymorphism (DNP) -   9. Reference sequence: Sequence of human reference genome in target     locus -   10. Mutation sequence: Sequence of cancer cell line genome in target     locus -   11. Amino acid change: Amino acid change due to nucleotide mutation -   12. Cancer cell line name: A name of cancer cell line which has     mutation -   13. Tissue of origin of tumor: Tissue from which cancer cell line     originated or was first separated from -   14. Ensembl transcript ID (ENST ID): Target gene transcript     identifier from Ensembl genomes database -   15. crRNA sequence: Extracted crRNA sequence from target mutation     locus of cancer cell line. PAM sequence is included in and     sequence's length varies depending on input crRNA length -   16. Distance between mutation and PAM: Distance from mutation to PAM     in crRNA. If PAM is upstream of crRNA, PAM's last position will be     position 0, mutation in PAM will be position negative, and mutation     in target cleavage site will be position positive.

In case of Cas9, PAM is on downstream of crRNA. So PAM's first position is position 0, mutation in PAM is position negative and mutation in target cleavage site is position positive.

-   17. Length of mutation: Length difference between reference sequence     and mutation sequence -   18. Reference target sequence: ±10 nucleotide sequence from     reference sequence of target locus -   19. Mutation target sequence: ±10 nucleotide sequence from cancer     cell line mutation sequence of target locus -   20. GuideRNA sequence (Tables 5a and 6): gRNA sequence with handle     and T7 RNA polymerase promoter sequences     -   GuideRNA sequence (Tables 5b and 7): gRNA sequence with handle,         tracrRNA and T7 RNA polymerase promoter sequences

Table 5a, below, shows illustrative output information obtained using this example for use with a Cas12a nuclease. Variations from a reference sequence (e.g., wild type, healthy, or non-cancerous) are shown in underlined text.

TABLE 5a GENE_ID 1956 4297 7157 GENE_SYMBOL EGFR KMT2A TP53 GRCH 37 37 37 CHR 7 11 17 MUT_START 55242465 118354982 7578205 MUT_END 55242479 118354983 7578205 MUT_CLASS In_Frame_Del Frame_Shift_ Missense_ Ins Mutation MUT_TYPE DEL INS SNP REF_SEQ GGAATTAAGAG — C AAGC (SEQ ID NO: 86) MUT_SEQ — A A AA_CHANGE p.ELREA746del p.Q1391fs p.S215I (“ELREA” disclosed as SEQ ID NO: 87) CELL_LINE NCIH1650 NCIH2126 NCIH661 TISSUE_ORI LUNG LUNG LUNG ENST_ID ENST00000275493.2 ENST00000389506.5 ENST00000269305.4 CRRNA_SEQ TTTGATAGCGAC TTTTTTCTTAGAAC TTTCGACATATTGT GGGAATTTTAAC TATTGCCATTGGAG GGTGGTGCCCTAT TTTC (SEQ ID (SEQ ID NO: 89) G (SEQ ID NO: 90) NO: 88) PAM_DIST −2 2 7 MUT_LEN 15 1 0 REF_TARGET TCGCTATCAAGG TAGTTCTAAGAAA CACCACCACACTA AATTAAGAGAA AA-TTCCA (SEQ ID TGTCGAAA (SEQ GCAACATCTCCG NO: 92) ID NO: 93) (SEQ ID NO: 91) MUT_TARGET TCGCTATCAA TAGTTCTAAGAAA CACCACCACAATA

AAATTCCA (SEQ TGTCGAAA (SEQ AACATCTCCG ID NO: 95) ID NO: 96) (SEQ ID NO: 94) GRNA AATTCTAATACG AATTCTAATACGAC AATTCTAATACGAC ACTCACTATAGgt TCACTATAGgtaatttct TCACTATAGgt Aatttctactaa actaagtgtagatTTCTTA aatttctactaa gtgtagat GAACTATTGCCATT gtgtagatGACAT ATAGCGACGGG GGAG (SEQ ID NO: ATTGTGGTGGTGCC AATTTTAACTTT 98) CTATG C (SEQ ID NO: 97) (SEQ ID NO: 99)

Table 5b, below, shows illustrative output information obtained using this example for use with a Cas9 nuclease. Variations from a reference sequence (e.g., wild type, healthy, or non-cancerous) are shown in underlined text.

TABLE 5b Output example (SpCas9) GENE_ID 1956 4297 7157 GENE_SYMBOL EGFR KMT2A TP53 GRCH 37 37 37 CHR 7 11 17 MUT_START 55242466 118354982 7577557 MUT_END 55242480 118354983 7577557 MUT_CLASS In_Frame_ Frame_Shift_ Missense_ Del Ins Mutation MUT_TYPE DEL INS SNP REF_SEQ GAATTAAGAGAA A GCA (SEQ ID NO: 100) MUT_SEQ - A G AA_CHANGE p.ELREA746del p.Q1391fs p.C242R (“ELREA” disclosed as SEQ ID NO: 87) CELL_LINE HCC827 NCIH2126 CFPAC1 TISSUE_ORI LUNG LUNG PANCREAS ENST_ID ENST00000275493.2 ENST00000389506.5 ENST00000269305.4 CRRNA_SEQ CGGAGATGTCTTG TAAGAAAAAATTC TCCGGTTCATGCC ATAGCGACGG CAGCAGATGG GCCCATGCGG (SEQ ID NO: 101) (SEQ ID NO: 102) (SEQ ID NO: 103) PAM_DIST 12 16 −1 MUT_LEN 15 1 0 REF_TARGET CGCTATCAAGGAA TAGTTCTAAG- CCGCCCATGCAGG TTAAGAGAAGCAA AAAAATTCCA AACTGTTA CATCTCCGA (SEQ (SEQ ID NO: 105) SEQ ID NO: 104) ID NO: 106) MUT_TARGET CGCTATCAAG TAGTTCTAAGAAA CCGCCCATGCGGG

AAATTCCA (SEQ AACTGTTA ACATCTCCGA ID NO: 95) (SEQ (SEQ ID NO: 107) ID NO: 108) GRNA attctaatacg attctaatacgac attctaatacgac actcactatag tcactatag tcactatag gCGGAGATGTCT gTAAGAAAAAATT gTCCGGTTCATG TGATAGCGAgtt CCAGCAGAgtttt CCGCCCATGgtt ttagagctagaa agagctagaaata ttagagctagaa atagcaagttaaa gcaagttaaaata atagcaagttaa ataaggctagtc aggctagtccgtt aataaggctagt cgttatcaac atcaacttgaaaa ccgttatcaact ttgaaaaagtgg agtggcaccgagt tgaaaaagtggc caccgagtc cggtgc accgagtcggtgc ggtgc (SEQ ID NO: (SEQ ID (SEQ ID NO: 110) NO: 111) 109)

Example 11. Guide Nucleic Acids for Targeting Cancer

A number of guide RNA sequences are consistent with the disclosure herein.

Table 6 and paragraph [00303] lists illustrative guide RNA sequences, determined using the methods described in Example 10, which can be used to guide a polypeptide comprising a Cas12a nuclease domain (e.g., a chimeric polypeptide comprising a Cas12a nuclease) to a target nucleic acid associated with a cancer, for example, lung cancer or pancreatic cancer. Variations from a reference sequence (e.g., wild type, healthy, or non-cancerous) are shown in underlined text.

TABLE 6 Illustrative guide RNA sequences for Cas12 comprising poylpeptide CRISPR gRNA ID: GF-CCELg12-1: [crRNA sequence]: crRNA sequence: T TTCCTCACTGGCTTCAGCGATTCCACA (SEQ ID NO: 112): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTCACTGGCTTCAGCGATTCCACA (SEQ ID NO: 113): [Target gene information]: Gene ID: 29123: Symbol: ANKRD11: Ensembl_Transcript_ID: ENST00000301030.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 89351168: mut_end: 89351168: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.K594K: mutation info source: CCLE: ref_target(−10 + 10): GCTCCTGCCT C TTCCTCACTG (SEQ ID NO: 114): mut_target(−10 + 10): GCTCCTGCCT T TTCCTCACTG (SEQ ID NO: 115): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-2: [crRNA sequence]: crRNA sequence: T T TTCCTCACTGGCTTCAGCGATTCCAC (SEQ ID NO: 116): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTCACTGGCTTCAGCGATTCCAC (SEQ ID NO: 117): [Target gene information]: Gene ID: 29123: Symbol: ANKRD11: Ensembl_Transcript_ID: ENST00000301030.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 89351168: mut_end: 89351168: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.K594K: mutation info source: CCLE: ref_target(−10 + 10): GCTCCTGCCT C TTCCTCACTG (SEQ ID NO: 114): mut_target(−10 + 10): GCTCCTGCCT T TTCCTCACTG (SEQ ID NO: 115): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-3: [crRNA sequence]: crRNA sequence: TTTCTTCAGGA A CGAAATCTCCCTCCAA (SEQ ID NO: 118): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCAGGAACGAAATCTCCCTCCAA (SEQ ID NO: 119): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112175363: mut_end: 112175363: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.A1358T: mutation info source: CCLE: ref_target(−10 + 10): TTCTTCAGGA G CGAAATCTCC (SEQ ID NO: 120): mut_target(−10 + 10): TTCTTCAGGA A CGAAATCTCC (SEQ ID NO: 121): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-4: [crRNA sequence]: crRNA sequence: TTTTCTTCAGGA A CGAAATCTCCCTCCA (SEQ ID NO: 122): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTCAGGAACGAAATCTCCCTCCA (SEQ ID NO: 123): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112175363: mut_end: 112175363: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.A1358T: mutation info source: CCLE: ref_target(−10 + 10): TTCTTCAGGA G CGAAATCTCC (SEQ ID NO: 120): mut_target(−10 + 10): TTCTTCAGGA A CGAAATCTCC (SEQ ID NO: 121): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-5: [crRNA sequence]: crRNA sequence: TTTCG T TCCTGAAGAAAATTCAACAGCT (SEQ ID NO: 124): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTCCTGAAGAAAATTCAACAGCT (SEQ ID NO: 125): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112175363: mut_end: 112175363: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.A1358T: mutation info source: CCLE: ref_target(−10 + 10): TTCTTCAGGA G CGAAATCTCC (SEQ ID NO: 120): mut_target(−10 + 10): TTCTTCAGGA A CGAAATCTCC (SEQ ID NO: 121): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-6: [crRNA sequence]: crRNA sequence: TTTGGAGGGAGATTTCG T TCCTGAAGAA (SEQ ID NO: 126): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGGGAGATTTCGTTCCTGAAGAA (SEQ ID NO: 127): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112175363: mut_end: 112175363: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.A1358T: mutation info source: CCLE: ref_target(−10 + 10): TTCTTCAGGA G CGAAATCTCC (SEQ ID NO: 120): mut_target(−10 + 10): TTCTTCAGGA A CGAAATCTCC (SEQ ID NO: 121): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-7: [crRNA sequence]: crRNA sequence: TTTTGGAGGGAGATTTCG T TCCTGAAGA (SEQ ID NO: 128): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGAGGGAGATTTCGTTCCTGAAGA (SEQ ID NO: 129): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112175363: mut_end: 112175363: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.A1358T: mutation info source: CCLE: ref_target(−10 + 10): TTCTTCAGGA G CGAAATCTCC (SEQ ID NO: 120): mut_target(−10 + 10): TTCTTCAGGA A CGAAATCTCC (SEQ ID NO: 121): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-8: [crRNA sequence]: crRNA sequence: TTTCAGGGAAAGGATG C AATCTGAATCA (SEQ ID NO: 130): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGGAAAGGATGCAATCTGAATCA (SEQ ID NO: 131): [Target gene information]: Gene ID: 324: Symbol: APC: Ensembl_ Transcript_ID: ENST00000457016.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 112177692: mut_end: 112177692: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S2134C: mutation info source: CCLE: ref_target(−10 + 10): GATTCAGATT C CATCCTTTCC (SEQ ID NO: 132): mut_target(−10 + 10): GATTCAGATT G CATCCTTTCC (SEQ ID NO: 133): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-9: [crRNA sequence]: crRNA sequence: TTTGAGACACCTGGGTCCATT A TTGTCA (SEQ ID NO: 134): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGACACCTGGGTCCATTATTGTCA (SEQ ID NO: 135): [Target gene information]: Gene ID: 171023: Symbol: ASXL1: Ensembl_ Transcript_ID: ENST00000375687.4: GRCh: 37: Chr: 20: [Target cancer mutation information]: mut_start: 31019180: mut_end: 31019180: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.L259I: mutation info source: CCLE: ref_target(−10 + 10): TGGGTCCATT C TTGTCAACAC (SEQ ID NO: 136): mut_target(−10 + 10): TGGGTCCATT A TTGTCAACAC (SEQ ID NO: 137): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-10: [crRNA sequence]: crRNA sequence: TTTTGAGACACCTGGGTCCATT A TTGTC (SEQ ID NO: 138): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGACACCTGGGTCCATTATTGTC (SEQ ID NO: 139): [Target gene information]: Gene ID: 171023: Symbol: ASXL1: Ensembl_ Transcript_ID: ENST00000375687.4: GRCh: 37: Chr: 20: [Target cancer mutation information]: mut_start: 31019180: mut_end: 31019180: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.L259I: mutation info source: CCLE: ref_target(−10 + 10): TGGGTCCATT C TTGTCAACAC (SEQ ID NO: 136): mut_target(−10 + 10): TGGGTCCATT A TTGTCAACAC (SEQ ID NO: 137): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-11: [crRNA sequence]: crRNA sequence: T TT GGGTAGGAGACTGTTTGATTCCTGG (SEQ ID NO: 140): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGTAGGAGACTGTTTGATTCCTGG (SEQ ID NO: 141): [Target gene information]: Gene ID: 171023: Symbol: ASXL1: Ensembl_ Transcript_ID: ENST00000375687.4: GRCh: 37: Chr: 20: [Target cancer mutation information]: mut_start: 31021717: mut_end: 31021718: mut_class: Missense_Mutation: mut_type: DNP: ref_seq: CC: mut_seq: TT: mut_aa: p.R573W: mutation info source: CCLE: ref_target(−10 + 10): TCCCGCCCAT CC GGGTAGGAGA (SEQ ID NO: 142): mut_target(−10 + 10): TCCCGCCCAT TT GGGTAGGAGA (SEQ ID NO: 143): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-12: [crRNA sequence]: crRNA sequence: TTTGCTCCATGGGAACATCTTTGCCTT A (SEQ ID NO: 144): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTCCATGGGAACATCTTTGCCTTA (SEQ ID NO: 145): [Target gene information]: Gene ID: 55252: Symbol: ASXL2: Ensembl_ Transcript_ID: ENST00000435504.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 25966334: mut_end: 25966334: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q958*: mutation info source: CCLE: ref_target(−10 + 10): TCTTTGCCTT G AAGCAGCTGA (SEQ ID NO: 146): mut_target(−10 + 10): TCTTTGCCTT A AAGCAGCTGA (SEQ ID NO: 147): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-13: [crRNA sequence]: crRNA sequence: TTTGCCTT A AAGCAGCTGAGTCACTAAA (SEQ ID NO: 148): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTTAAAGCAGCTGAGTCACTAAA (SEQ ID NO: 149): [Target gene information]: Gene ID: 55252: Symbol: ASXL2: Ensembl_ Transcript_ID: ENST00000435504.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 25966334: mut_end: 25966334: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q958*: mutation info source: CCLE: ref_target(−10 + 10): TCTTTGCCTT G AAGCAGCTGA (SEQ ID NO: 146): mut_target(−10 + 10): TCTTTGCCTT A AAGCAGCTGA (SEQ ID NO: 147): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 5: indel length: 0:: CRISPR gRNA ID: GF-CCELg12-14: [crRNA sequence]: crRNA sequence: TTTAGTGACTCAGCTGCTT T AAGGCAAA (SEQ ID NO: 150): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTGACTCAGCTGCTTTAAGGCAAA (SEQ ID NO: 151): [Target gene information]: Gene ID: 55252: Symbol: ASXL2: Ensembl_ Transcript_ID: ENST00000435504.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 25966334: mut_end: 25966334: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q958*: mutation info source: CCLE: ref_target(−10 + 10): TCTTTGCCTT G AAGCAGCTGA (SEQ ID NO: 146): mut_target(−10 + 10): TCTTTGCCTT A AAGCAGCTGA (SEQ ID NO: 147): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-15: [crRNA sequence]: crRNA sequence: TT T AAGGCAAAGATGTTCCCATGGAGCA (SEQ ID NO: 152): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGCAAAGATGTTCCCATGGAGCA (SEQ ID NO: 153): [Target gene information]: Gene ID: 55252: Symbol: ASXL2: Ensembl_ Transcript_ID: ENST00000435504.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 25966334: mut_end: 25966334: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q958*: mutation info source: CCLE: ref_target(−10 + 10): TCTTTGCCTT G AAGCAGCTGA (SEQ ID NO: 146): mut_target(−10 + 10): TCTTTGCCTT A AAGCAGCTGA (SEQ ID NO: 147): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-16: [crRNA sequence]: crRNA sequence: TTTCTGTGAGAAA G TACTGGTGTGAAAT (SEQ ID NO: 154): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTGAGAAAGTACTGGTGTGAAAT (SEQ ID NO: 155): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108106527: mut_end: 108106527: mut_class: Silent: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.K154K: mutation info source: CCLE: ref_target(−10 + 10): CTGTGAGAAA A TACTGGTGTG (SEQ ID NO: 156): mut_target(−10 + 10): CTGTGAGAAA G TACTGGTGTG (SEQ ID NO: 157): [Model Cell line information]: cell: SW1990: cancer_type: PANCREAS: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF- CCELg12-17: [crRNA sequence]: crRNA sequence: TTTCACACCAGTA C TTTCTCACAGAAAG (SEQ ID NO: 158): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACACCAGTACTTTCTCACAGAAAG (SEQ ID NO: 159): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108106527: mut_end: 108106527: mut_class: Silent: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.K154K: mutation info source: CCLE: ref_target(−10 + 10): CTGTGAGAAA A TACTGGTGTG (SEQ ID NO: 156): mut_target(−10 + 10): CTGTGAGAAA G TACTGGTGTG (SEQ ID NO: 157): [Model Cell line information]: cell: SW1990: cancer_type: PANCREAS: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF- CCELg12-18: [crRNA sequence]: crRNA sequence: TTTCTTGAAGATTTTTACA A AAATATAT (SEQ ID NO: 160): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGAAGATTTTTACAAAAATATAT (SEQ ID NO: 161): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-19: [crRNA sequence]: crRNA sequence: TTTGTTTTCTTGAAGATTTTTACA A AAA (SEQ ID NO: 164): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTCTTGAAGATTTTTACAAAAA (SEQ ID NO: 165): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-20: [crRNA sequence]: crRNA sequence: TTTACA A AAATATATTCAGAAAGAAACA (SEQ ID NO: 166): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAAAAATATATTCAGAAAGAAACA (SEQ ID NO: 167): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-21: [crRNA sequence]: crRNA sequence: TTTTGTTTTCTTGAAGATTTTTACA A AA (SEQ ID NO: 168): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTTTCTTGAAGATTTTTACAAAA (SEQ ID NO: 169): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-22: [crRNA sequence]: crRNA sequence: TTTTCTTGAAGATTTTTACA A AAATATA (SEQ ID NO: 170): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGAAGATTTTTACAAAAATATA (SEQ ID NO: 171): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-23: [crRNA sequence]: crRNA sequence: TTTTTACA A AAATATATTCAGAAAGAAA (SEQ ID NO: 172): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TACAAAAATATATTCAGAAAGAAA (SEQ ID NO: 173): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-24: [crRNA sequence]: crRNA sequence: TTTTACA A AAATATATTCAGAAAGAAAC (SEQ ID NO: 174): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACAAAAATATATTCAGAAAGAAAC (SEQ ID NO: 175): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-25: [crRNA sequence]: crRNA sequence: TTTCTTTCTGAATATATTT T TGTAAAAA (SEQ ID NO: 176): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCTGAATATATTTTTGTAAAAA (SEQ ID NO: 177): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-26: [crRNA sequence]: crRNA sequence: TTTCTGAATATATTT T TGTAAAAATCTT (SEQ ID NO: 178): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGAATATATTTTTGTAAAAATCTT (SEQ ID NO: 179): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-27: [crRNA sequence]: crRNA sequence: T T TGTAAAAATCTTCAAGAAAACAAAAT (SEQ ID NO: 180): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAAAAATCTTCAAGAAAACAAAAT (SEQ ID NO: 181): [Target gene information]: Gene ID: 472: Symbol: ATM Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-28: [crRNA sequence]: crRNA sequence: TTT T TGTAAAAATCTTCAAGAAAACAAA (SEQ ID NO: 182): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTAAAAATCTTCAAGAAAACAAA (SEQ ID NO: 183): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-29: [crRNA sequence]: crRNA sequence: TT T TGTAAAAATCTTCAAGAAAACAAAA (SEQ ID NO: 184): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTAAAAATCTTCAAGAAAACAAAA (SEQ ID NO: 185): [Target gene information]: Gene ID: 472: Symbol: ATM: Ensembl_ Transcript_ID: ENST00000452508.2: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 108099914: mut_end: 108099914: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q65Q: mutation info source: CCLE: ref_target(−10 + 10): GATTTTTACA G AAATATATTC (SEQ ID NO: 162): mut_target(−10 + 10): GATTTTTACA A AAATATATTC (SEQ ID NO: 163): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-30: [crRNA sequence]: crRNA sequence: TTTGCGGCC T TAAAATTAAAAACAACAT (SEQ ID NO: 186): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGGCCTTAAAATTAAAAACAACAT (SEQ ID NO: 187): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-31: [crRNA sequence]: crRNA sequence: TTTTGCGGCC T TAAAATTAAAAACAACA (SEQ ID NO: 190): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCGGCCTTAAAATTAAAAACAACA (SEQ ID NO: 191): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-32: [crRNA sequence]: crRNA sequence: TTTAATTTTA A GGCCGCAAAAGGAGATT (SEQ ID NO: 192): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTTTAAGGCCGCAAAAGGAGATT (SEQ ID NO: 193): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-33: [crRNA sequence]: crRNA sequence: TTTA A GGCCGCAAAAGGAGATTTGGTAC (SEQ ID NO: 194): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGCCGCAAAAGGAGATTTGGTAC (SEQ ID NO: 195): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-34: [crRNA sequence]: crRNA sequence: TTTTTAATTTTA A GGCCGCAAAAGGAGA (SEQ ID NO: 196): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAATTTTAAGGCCGCAAAAGGAGA (SEQ ID NO: 197): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-35: [crRNA sequence]: crRNA sequence: TTTTAATTTTA A GGCCGCAAAAGGAGAT (SEQ ID NO: 198): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATTTTAAGGCCGCAAAAGGAGAT (SEQ ID NO: 199): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-36: [crRNA sequence]: crRNA sequence: TTTTA A GGCCGCAAAAGGAGATTTGGTA (SEQ ID NO: 200): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGGCCGCAAAAGGAGATTTGGTA (SEQ ID NO: 201): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142272241: mut_end: 142272241: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): TTTTGCGGCC C TAAAATTAAA (SEQ ID NO: 188): mut_target(−10 + 10): TTTTGCGGCC T TAAAATTAAA (SEQ ID NO: 189): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-37: [crRNA sequence]: crRNA sequence: T T TCCTGCCCAGGCATCCTCCTATTTTT (SEQ ID NO: 202): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGCCCAGGCATCCTCCTATTTTT (SEQ ID NO: 203): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142178136: mut_end: 142178136: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L2428F: mutation info source: CCLE: ref_target(−10 + 10): CTGGGCAGGA G AAATTCTCGG (SEQ ID NO: 204): mut_target(−10 + 10): CTGGGCAGGA A AAATTCTCGG (SEQ ID NO: 205): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-38: [crRNA sequence]: crRNA sequence: TTT T TCCTGCCCAGGCATCCTCCTATTT (SEQ ID NO: 206): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCTGCCCAGGCATCCTCCTATTT (SEQ ID NO: 207): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142178136: mut_end: 142178136: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L2428F: mutation info source: CCLE: ref_target(−10 + 10): CTGGGCAGGA G AAATTCTCGG (SEQ ID NO: 204): mut_target(−10 + 10): CTGGGCAGGA A AAATTCTCGG (SEQ ID NO: 205): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-39: [crRNA sequence]: crRNA sequence: TT T TCCTGCCCAGGCATCCTCCTATTTT (SEQ ID NO: 208): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTGCCCAGGCATCCTCCTATTTT (SEQ ID NO: 209): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142178136: mut_end: 142178136: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L2428F: mutation info source: CCLE: ref_target(−10 + 10): CTGGGCAGGA G AAATTCTCGG (SEQ ID NO: 204): mut_target(−10 + 10): CTGGGCAGGA A AAATTCTCGG (SEQ ID NO: 205): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-40: [crRNA sequence]: crRNA sequence: TTTC C AATTGCACTGACTCCGGCCACTC (SEQ ID NO: 210): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAATTGCACTGACTCCGGCCACTC (SEQ ID NO: 211): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142224091: mut_end: 142224091: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.R1696G: mutation info source: CCLE: ref_target(−10 + 10): TCTGCCTTTC T AATTGCACTG (SEQ ID NO: 212): mut_target(−10 + 10): TCTGCCTTTC C AATTGCACTG (SEQ ID NO: 213): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-41: [crRNA sequence]: crRNA sequence: TTTAGAGATGGTTCTGCCTTTC C AATTG (SEQ ID NO: 214): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGATGGTTCTGCCTTTCCAATTG (SEQ ID NO: 215): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142224091: mut_end: 142224091: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.R1696G: mutation info source: CCLE: ref_target(−10 + 10): TCTGCCTTTC T AATTGCACTG (SEQ ID NO: 212): mut_target(−10 + 10): TCTGCCTTTC C AATTGCACTG (SEQ ID NO: 213): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-42: [crRNA sequence]: crRNA sequence: TTTTAGAGATGGTTCTGCCTTTC C AATT (SEQ ID NO: 216): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGAGATGGTTCTGCCTTTCCAATT (SEQ ID NO: 217): [Target gene information]: Gene ID: 545: Symbol: ATR: Ensembl_ Transcript_ID: ENST00000350721.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 142224091: mut_end: 142224091: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.R1696G: mutation info source: CCLE: ref_target(−10 + 10): TCTGCCTTTC T AATTGCACTG (SEQ ID NO: 212): mut_target(−10 + 10): TCTGCCTTTC C AATTGCACTG (SEQ ID NO: 213): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-43: [crRNA sequence]: crRNA sequence: TTTCC C AGATTTTCAGATGTTAAGTAGA (SEQ ID NO: 218): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCAGATTTTCAGATGTTAAGTAGA (SEQ ID NO: 219): [Target gene information]: Gene ID: 546: Symbol: ATRX: Ensembl_ Transcript_ID: ENST00000373344.5: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 76855979: mut_end: 76855979: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.Q1874P: mutation info source: CCLE: ref_target(−10 + 10): CTGAAAATCT T GGAAAAGCTT (SEQ ID NO: 220): mut_target(−10 + 10): CTGAAAATCT G GGAAAAGCTT (SEQ ID NO: 221): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-44: [crRNA sequence]: crRNA sequence: TTTTCC C AGATTTTCAGATGTTAAGTAG (SEQ ID NO: 222): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCCAGATTTTCAGATGTTAAGTAG (SEQ ID NO: 223): [Target gene information]: Gene ID: 546: Symbol: ATRX: Ensembl_ Transcript_ID: ENST00000373344.5: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 76855979: mut_end: 76855979: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.Q1874P: mutation info source: CCLE: ref_target(−10 + 10): CTGAAAATCT T GGAAAAGCTT (SEQ ID NO: 220): mut_target(−10 + 10): CTGAAAATCT G GGAAAAGCTT (SEQ ID NO: 221): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-45: [crRNA sequence]: crRNA sequence:  T TTTTTTGCCTTCTTAATCATCTCTTTG (SEQ ID NO: 224): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTGCCTTCTTAATCATCTCTTTG (SEQ ID NO: 225): [Target gene information]: Gene ID: 546: Symbol: ATRX: Ensembl_ Transcript_ID: ENST00000373344.5: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 76939673: mut_end: 76939674: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: T: mut_aa: p.L359fs: mutation info source: CCLE: ref_target(−10 + 10): GTCTCAATCA-TTTTTTGCCT (SEQ ID NO: 226): mut_target(−10 + 10): GTCTCAATCA T TTTTTTGCCT (SEQ ID NO: 227): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_dist: −3: indel length: 1: CRISPR gRNA ID:  GF-CCELg12-46: [crRNA sequence]: crRNA sequence: T T TTTTTCCCCTTTTTCCCTTTTTTCTT (SEQ ID NO: 228): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCCCCTTTTTCCCTTTTTTCTT (SEQ ID NO: 229): [Target gene information]: Gene ID: 546: Symbol: ATRX: Ensembl_ Transcript_ID: ENST00000373344.5: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 76855018: mut_end: 76855019: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: T: mut_aa: p.D1940fs: mutation info source: CCLE: ref_target(-10 + 10): GAGCTACTAT-TTTTTCCCCT (SEQ ID NO: 230): mut_target(−10 + 10): GAGCTACTAT T TTTTTCCCCT (SEQ ID NO: 231): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −2: indel length: 1: CRISPR gRNA ID: GF-CCELg12-47: [crRNA sequence]: crRNA sequence:  T TTTTTCCCCTTTTTCCCTTTTTTCTTC (SEQ ID NO: 232): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCCCCTTTTTCCCTTTTTTCTTC (SEQ ID NO: 233): [Target gene information]: Gene ID: 546: Symbol: ATRX: Ensembl_ Transcript_ID: ENST00000373344.5: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 76855018: mut_end: 76855019: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: T: mut_aa: p.D1940fs: mutation info source: CCLE: ref_target(−10 + 10): GAGCTACTAT-TTTTTCCCCT (SEQ ID NO: 230): mut_target(−10 + 10): GAGCTACTAT T TTTTTCCCCT (SEQ ID NO: 231): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −3: indel length: 1: CRISPR gRNA ID: GF-CCELg12-48: [crRNA sequence]: crRNA sequence: TTTGGGCAACGAGAC A GATCCTCATCAG (SEQ ID NO: 235): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGCAACGAGACAGATCCTCATCAG (SEQ ID NO: 236): [Target gene information]: Gene ID: 673: Symbol: BRAF: Ensembl_ Transcript_ID: ENST00000288602.6: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 140494164: mut_end: 140494164: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R362R: mutation info source: CCLE: ref_target(−10 + 10): GATGAGGATC G GTCTCGTTGC (SEQ ID NO: 237): mut_target(−10 + 10): GATGAGGATC T GTCTCGTTGC (SEQ ID NO: 238): [Model Cell line information]: cell: NCIH661: cancer type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-49: [crRNA sequence]: crRNA sequence: TTTGGCCAACAATACACAC A TTTTTCTG (SEQ ID NO: 239): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCCAACAATACACACATTTTTCTG (SEQ ID NO: 240): [Target gene information]: Gene ID: 672: Symbol: BRCA1: Ensembl_ Transcript_ID: ENST00000357654.3: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 41234421: mut_end: 41234421: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A1453S: mutation info source: CCLE: ref_target(−10 + 10): CAATACACAC C TTTTTCTGAT (SEQ ID NO: 241): mut_target(−10 + 10): CAATACACAC A TTTTTCTGAT (SEQ ID NO: 242): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-50: [crRNA sequence]: crRNA sequence: TTTCCACT G CCTGTGGGACTCTCCAACA (SEQ ID NO: 243): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CACTGCCTGTGGGACTCTCCAACA (SEQ ID NO: 244): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59937215: mut_end: 59937215: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.G49G: mutation info source: CCLE: ref_target(−10 + 10): TTTTTCCACT T CCTGTGGGAC (SEQ ID NO: 245): mut_target(−10 + 10): TTTTTCCACT G CCTGTGGGAC (SEQ ID NO: 246): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-51: [crRNA sequence]: crRNA sequence: TTTTTCCACT G CCTGTGGGACTCTCCAA (SEQ ID NO: 247): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCACTGCCTGTGGGACTCTCCAA (SEQ ID NO: 248): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_ Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59937215: mut_end: 59937215: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.G49G: mutation info source: CCLE: ref_target(−10 + 10): TTTTTCCACT T CCTGTGGGAC (SEQ ID NO: 245): mut_target(−10 + 10): TTTTTCCACT G CCTGTGGGAC (SEQ ID NO: 246): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-52: [crRNA sequence]: crRNA sequence: TTTTCCACT G CCTGTGGGACTCTCCAAC (SEQ ID NO: 249): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCACTGCCTGTGGGACTCTCCAAC (SEQ ID NO: 250): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_ Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59937215: mut_end: 59937215: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.G49G: mutation info source: CCLE: ref_target(−10 + 10): TTTTTCCACT T CCTGTGGGAC (SEQ ID NO: 245): mut_target(−10 + 10): TTTTTCCACT G CCTGTGGGAC (SEQ ID NO: 246): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-53: [crRNA sequence]: crRNA sequence: TTTGTTGGAGAGTCCCACAGG C AGTGGA (SEQ ID NO: 251): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGGAGAGTCCCACAGGCAGTGGA (SEQ ID NO: 252): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_ Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59937215: mut_end: 59937215: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.G49G: mutation info source: CCLE: ref_target(−10 + 10): TTTTTCCACT T CCTGTGGGAC (SEQ ID NO: 245): mut_target(−10 + 10): TTTTTCCACT G CCTGTGGGAC (SEQ ID NO: 246): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-54: [crRNA sequence]: crRNA sequence: TTTCAAAGGTTGAATGGTG CG AATCTGC (SEQ ID NO: 253): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAGGTTGAATGGTGCGAATCTGC (SEQ ID NO: 254): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_ Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59763496: mut_end: 59763496: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: T: mut_seq: -: mut_aa: p.Q869fs: mutation info source: CCLE: ref_target(−10 + 10): TGAATGGTGC T GAATCTGCTG (SEQ ID NO: 255): mut_target(−10 + 10): TGAATGGTGC-GAATCTGCTG (SEQ ID NO: 256): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 1: CRISPR gRNA ID: GF-CCELg12-55: [crRNA sequence]: crRNA sequence: TTTCTAAATGGGTACGGCAGCAGATT CG (SEQ ID NO: 257): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAAATGGGTACGGCAGCAGATTCG (SEQ ID NO: 258): [Target gene information]: Gene ID: 83990: Symbol: BRIP1: Ensembl_ Transcript_ID: ENST00000259008.2: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 59763496: mut_end: 59763496: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: T: mut_seq: -: mut_aa: p.Q869fs: mutation info source: CCLE: ref_target(−10 + 10): TGAATGGTGC T GAATCTGCTG (SEQ ID NO: 255): mut_target(−10 + 10): TGAATGGTGC-GAATCTGCTG (SEQ ID NO: 256): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 24: indel length: 1: CRISPR gRNA ID: GF-CCELg12-56: [crRNA sequence]: crRNA sequence: TTTGATCCTAGAGGG G GTGGCAGCCTGT (SEQ ID NO: 259): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATCCTAGAGGGGGTGGCAGCCTGT (SEQ ID NO: 260): [Target gene information]: Gene ID: 867: Symbol: CBL: Ensembl_ Transcript_ID: ENST00000264033.4: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 119149307: mut_end: 119149307: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.5439G: mutation info source: CCLE: ref_target(−10 + 10): TCCTAGAGGG A GTGGCAGCCT (SEQ ID NO: 261): mut_target(−10 + 10): TCCTAGAGGG G GTGGCAGCCT (SEQ ID NO: 262): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-57: [crRNA sequence]: crRNA sequence: TTTCACCGAAGGCCGG A ACCAGGGCAGC (SEQ ID NO: 263): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACCGAAGGCCGGAACCAGGGCAGC (SEQ ID NO: 264): [Target gene information]: Gene ID: 80381: Symbol: CD276: Ensembl_Transcript_ID: ENST00000318443.5: GRCh: 37: Chr: 15: [Target cancer mutation information]: mut_start: 73996170: mut_end: 73996170: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.D302N: mutation info source: CCLE: ref_target(−10 + 10): CGAAGGCCGG G ACCAGGGCAG (SEQ ID NO: 265): mut_target(−10 + 10): CGAAGGCCGG A ACCAGGGCAG (SEQ ID NO: 266): [Model Cell line information]: cell: NCIH1573: cancer type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-58: [crRNA sequence]: crRNA sequence: TTTGGAGGACAATAATA T ATGTCCCAAG (SEQ ID NO: 267): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGGACAATAATATATGTCCCAAG (SEQ ID NO: 268): [Target gene information]: Gene ID: 64326: Symbol: RFWD2: Ensembl_ Transcript_ID: ENST00000367669.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 176145105: mut_end: 176145105: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.R169I: mutation info source: CCLE: ref_target(−10 + 10): CTTGGGACAT C TATTATTGTC (SEQ ID NO: 269): mut_target(−10 + 10): CTTGGGACAT A TATTATTGTC (SEQ ID NO: 270): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-59: [crRNA sequence]: crRNA sequence: TTTATCCAATATT A TTTCTACTTCTTGT (SEQ ID NO: 271): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCAATATTATTTCTACTTCTTGT (SEQ ID NO: 272): [Target gene information]: Gene ID: 8452: Symbol: CUL3: Ensembl_ Transcript_ID: ENST00000264414.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 225368517: mut_end: 225368517: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.T410I: mutation info source: CCLE: ref_target(−10 + 10): ATCCAATATT G TTTCTACTTC (SEQ ID NO: 273): mut_target(−10 + 10): ATCCAATATT A TTTCTACTTC (SEQ ID NO: 274): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-60: [crRNA sequence]: crRNA sequence: TTTCAGGGACAA A TGTGCTGTGCTTACA (SEQ ID NO: 275): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGGACAAATGTGCTGTGCTTACA (SEQ ID NO: 276): [Target gene information]: Gene ID: 23405: Symbol: DICER1: Ensembl_Transcript_ID: ENST00000526495.1: GRCh: 37: Chr: 14: [Target cancer mutation information]: mut_start: 95570354: mut_end: 95570354: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.I1127F: mutation info source: CCLE: ref_target(−10 + 10): TCAGGGACAA T TGTGCTGTGC (SEQ ID NO: 277): mut_target(−10 + 10): TCAGGGACAA A TGTGCTGTGC (SEQ ID NO: 278): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-61: [crRNA sequence]: crRNA sequence: TTTTCAGGGACAA A TGTGCTGTGCTTAC (SEQ ID NO: 279): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGGGACAAATGTGCTGTGCTTAC (SEQ ID NO: 280): [Target gene information]: Gene ID: 23405: Symbol: DICER1: Ensembl_Transcript_ID: ENST00000526495.1: GRCh: 37: Chr: 14: [Target cancer mutation information]: mut_start: 95570354: mut_end: 95570354: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.I1127F: mutation info source: CCLE: ref_target(−10 + 10): TCAGGGACAA T TGTGCTGTGC (SEQ ID NO: 277): mut_target(−10 + 10): TCAGGGACAA A TGTGCTGTGC (SEQ ID NO: 278): [Model Cell line information]: cell: NCIH1975: cancer type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-62: [crRNA sequence]: crRNA sequence:  T TTGTCCCTGAAAATGCTGCACATCAAG (SEQ ID NO: 281): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCCTGAAAATGCTGCACATCAAG (SEQ ID NO: 282): [Target gene information]: Gene ID: 23405: Symbol: DICER1: Ensembl_Transcript_ID: ENST00000526495.1: GRCh: 37: Chr: 14: [Target cancer mutation information]: mut_start: 95570354: mut_end: 95570354: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.I1127F: mutation info source: CCLE: ref_target(−10 + 10): TCAGGGACAA T TGTGCTGTGC (SEQ ID NO: 277): mut_target(−10 + 10): TCAGGGACAA A TGTGCTGTGC (SEQ ID NO: 278): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-63: [crRNA sequence]: crRNA sequence: TT T GTCTGCTCACCAAGAAGTTCATTCA (SEQ ID NO: 283): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTGCTCACCAAGAAGTTCATTCA (SEQ ID NO: 284): [Target gene information]: Gene ID: 1871: Symbol: E2F3: Ensembl_ Transcript_ID: ENST00000346618.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 20481481: mut_end: 20481481: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G184C: mutation info source: CCLE: ref_target(−10 + 10): TACGTCTCTT G GTCTGCTCAC (SEQ ID NO: 285): mut_target(−10 + 10): TACGTCTCTT T GTCTGCTCAC (SEQ ID NO: 286): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-64: [crRNA sequence]: crRNA sequence: TTTGGGC G GGCCAAACTGCTGGGTGCGG (SEQ ID NO: 287): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGCGGGCCAAACTGCTGGGTGCGG (SEQ ID NO: 288): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55259515: mut_end: 55259515: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.L858R: mutation info source: CCLE: ref_target(−10 + 10): GATTTTGGGC T GGCCAAACTG (SEQ ID NO: 289): mut_target(−10 + 10): GATTTTGGGC G GGCCAAACTG (SEQ ID NO: 290): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-65: [crRNA sequence]: crRNA sequence: TTTTGGGC G GGCCAAACTGCTGGGTGCG (SEQ ID NO: 291): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGGCGGGCCAAACTGCTGGGTGCG (SEQ ID NO: 292): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55259515: mut_end: 55259515: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.L858R: mutation info source: CCLE: ref_target(−10 + 10): GATTTTGGGC T GGCCAAACTG (SEQ ID NO: 289): mut_target(−10 + 10): GATTTTGGGC G GGCCAAACTG (SEQ ID NO: 290): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-66: [crRNA sequence]: crRNA sequence: TTTGGCC C GCCCAAAATCTGTGATCTTG (SEQ ID NO: 293): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCCCGCCCAAAATCTGTGATCTTG (SEQ ID NO: 294): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55259515: mut_end: 55259515: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.L858R: mutation info source: CCLE: ref_target(−10 + 10): GATTTTGGGC T GGCCAAACTG (SEQ ID NO: 289): mut_target(−10 + 10): GATTTTGGGC G GGCCAAACTG (SEQ ID NO: 290): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-67: [crRNA sequence]: crRNA sequence: TTTCCTTGTTGGCTTTCGGAGATGT TT T (SEQ ID NO: 295): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGTTGGCTTTCGGAGATGTTTT (SEQ ID NO: 296): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242465: mut_end: 55242479: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GGAATTAAGAGAAGC (SEQ ID NO: 86): mut_seq: -: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): TCGCTATCAA GGAATTAAGAGAAGC AACATCTCCG (SEQ ID NO: 91): mut_target(−10 + 10): TCGCTATCAA

AACATCTCCG (SEQ ID NO: 94): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 23: indel length: 15: CRISPR gRNA ID: GF-CCELg12-68: [crRNA sequence]: crRNA sequence: TTTCGGAGATGT TT TGATAGCGACGGGA (SEQ ID NO: 297): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGAGATGTTTTGATAGCGACGGGA (SEQ ID NO: 298): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242465: mut_end: 55242479: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GGAATTAAGAGAAGC (SEQ ID NO: 86): mut_seq:-: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): TCGCTATCAA GGAATTAAGAGAAGC AACATCTCCG (SEQ ID NO: 91): mut_target(−10 + 10): TCGCTATCAA

AACATCTCCG (SEQ ID NO: 94): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: 10: indel length: 15: CRISPR gRNA ID: GF-CCELg12-69: [crRNA sequence]: crRNA sequence:  TT TGATAGCGACGGGAATTTTAACTTTC (SEQ ID NO: 88): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATAGCGACGGGAATTTTAACTTTC (SEQ ID NO: 97): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242465: mut_end: 55242479: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GGAATTAAGAGAAGC (SEQ ID NO: 86): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): TCGCTATCAA GGAATTAAGAGAAGC AACATCTCCG (SEQ ID NO: 91): mut_target(−10 + 10): TCGCTATCAA

AACATCTCCG (SEQ ID NO: 94): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: −2: indel length: 15: CRISPR gRNA ID: GF-CCELg12-70: [crRNA sequence]: crRNA sequence: T TT TGATAGCGACGGGAATTTTAACTTT (SEQ ID NO: 299): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GATAGCGACGGGAATTTTAACTTT (SEQ ID NO: 300): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242465: mut_end: 55242479: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GGAATTAAGAGAAGC (SEQ ID NO: 86): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): TCGCTATCAA GGAATTAAGAGAAGC AACATCTCCG (SEQ ID NO: 91): mut_target(−10 + 10): TCGCTATCAA

AACATCTCCG (SEQ ID NO: 94): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG: PAM_dist: −1: indel length: 15: CRISPR gRNA ID: GF-CCELg12-71: [crRNA sequence]: crRNA sequence: TTTCCTTGTTGGCTTTCGGAGATG TC TT (SEQ ID NO: 301): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGTTGGCTTTCGGAGATGTCTT (SEQ ID NO: 302): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242466: mut_end: 55242480: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GAATTAAGAGAAGCA (SEQ ID NO: 100): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): CGCTATCAAG GAATTAAGAGAAGCA ACATCTCCGA (SEQ ID NO: 104): mut_target(-10 + 10): CGCTATCAAG

ACATCTCCGA (SEQ ID NO: 107): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: 22: indel length: 15: CRISPR gRNA ID: GF-CCELg12-72: [crRNA sequence]: crRNA sequence: TTTCGGAGATG TC TTGATAGCGACGGGA (SEQ ID NO: 303): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGAGATGTCTTGATAGCGACGGGA (SEQ ID NO: 304): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242466: mut_end: 55242480: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GAATTAAGAGAAGCA (SEQ ID NO: 100): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): CGCTATCAAG GAATTAAGAGAAGCA ACATCTCCGA (SEQ ID NO: 104): mut_target(−10 + 10): CGCTATCAAG

ACATCTCCGA (SEQ ID NO: 107): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: 9: indel length: 15: CRISPR gRNA ID: GF-CCELg12-73: [crRNA sequence]: crRNA sequence: TTTCCTTGTTGGCTTTCGGAGATG TC TT (SEQ ID NO: 301): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGTTGGCTTTCGGAGATGTCTT (SEQ ID NO: 302): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242466: mut_end: 55242480: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GAATTAAGAGAAGCA (SEQ ID NO: 100): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): CGCTATCAAG GAATTAAGAGAAGCA ACATCTCCGA (SEQ ID NO: 104): mut_target(−10 + 10): CGCTATCAAG

ACATCTCCGA (SEQ ID NO: 107): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 22: indel length: 15: CRISPR gRNA ID: GF-CCELg12-74: [crRNA sequence]: crRNA sequence: TTTCGGAGATG TC TTGATAGCGACGGGA (SEQ ID NO: 303): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGAGATGTCTTGATAGCGACGGGA (SEQ ID NO: 304): [Target gene information]: Gene ID: 1956: Symbol: EGFR: Ensembl_ Transcript_ID: ENST00000275493.2: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 55242466: mut_end: 55242480: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GAATTAAGAGAAGCA (SEQ ID NO: 100): mut_seq: −: mut_aa: p.ELREA746del (“ELREA” disclosed as SEQ ID NO: 87): mutation info source: CCLE: ref_target(−10 + 10): CGCTATCAAG GAATTAAGAGAAGCA ACATCTCCGA (SEQ ID NO: 104): mut_target(−10 + 10): CGCTATCAAG

ACATCTCCGA (SEQ ID NO: 107): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 9: indel length: 15: CRISPR gRNA ID: GF-CCELg12-75: [crRNA sequence]: crRNA sequence: TTTAAATTCTTTACAGTGCACTTC A AAA (SEQ ID NO: 305): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATTCTTTACAGTGCACTTCAAAA (SEQ ID NO: 306): [Target gene information]: Gene ID: 4072: Symbol: EPCAM: Ensembl_ Transcript_ID: ENST00000263735.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 47604162: mut_end: 47604162: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q167Q: mutation info source: CCLE: ref_target(−10 + 10): GTGCACTTCA G AAGGAGATCA (SEQ ID NO: 307): mut_target(−10 + 10): GTGCACTTCA A AAGGAGATCA (SEQ ID NO: 308): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-76: [crRNA sequence]: crRNA sequence: TTTACAGTGCACTTCA A AAGGAGATCAC (SEQ ID NO: 309): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGTGCACTTCAAAAGGAGATCAC (SEQ ID NO: 310): [Target gene information]: Gene ID: 4072: Symbol: EPCAM: Ensembl_ Transcript_ID: ENST00000263735.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 47604162: mut_end: 47604162: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q167Q: mutation info source: CCLE: ref_target(−10 + 10): GTGCACTTCA G AAGGAGATCA (SEQ ID NO: 307): mut_target(−10 + 10): GTGCACTTCA A AAGGAGATCA (SEQ ID NO: 308): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-77: [crRNA sequence]: crRNA sequence: TTTTAAATTCTTTACAGTGCACTTCA A A (SEQ ID NO: 311): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAATTCTTTACAGTGCACTTCAAA (SEQ ID NO: 312): [Target gene information]: Gene ID: 4072: Symbol: EPCAM: Ensembl_ Transcript_ID: ENST00000263735.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 47604162: mut_end: 47604162: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q167Q: mutation info source: CCLE: ref_target(−10 + 10): GTGCACTTCA G AAGGAGATCA (SEQ ID NO: 307): mut_target(−10 + 10): GTGCACTTCA A AAGGAGATCA (SEQ ID NO: 308): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-78: [crRNA sequence]: crRNA sequence: T T TGAAGTGCACTGTAAAGAATTTAAAA (SEQ ID NO: 313): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGTGCACTGTAAAGAATTTAAAA (SEQ ID NO: 314): [Target gene information]: Gene ID: 4072: Symbol: EPCAM: Ensembl_ Transcript_ID: ENST00000263735.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 47604162: mut_end: 47604162: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q167Q: mutation info source: CCLE: ref_target(−10 + 10): GTGCACTTCA G AAGGAGATCA (SEQ ID NO: 307): mut_target(−10 + 10): GTGCACTTCA A AAGGAGATCA (SEQ ID NO: 308): [Model Cell line information]: cell: NCIH1975: cancer type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-79: [crRNA sequence]: crRNA sequence: TT T TGAAGTGCACTGTAAAGAATTTAAA (SEQ ID NO: 315): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAAGTGCACTGTAAAGAATTTAAA (SEQ ID NO: 316): [Target gene information]: Gene ID: 4072: Symbol: EPCAM: Ensembl_ Transcript_ID: ENST00000263735.4: GRCh: 37: Chr: 2: [Target cancer mutation information]: mut_start: 47604162: mut_end: 47604162: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q167Q: mutation info source: CCLE: ref_target(−10 + 10): GTGCACTTCA G AAGGAGATCA (SEQ ID NO: 307): mut_target(−10 + 10): GTGCACTTCA A AAGGAGATCA (SEQ ID NO: 308): [Model Cell line information]: cell: NCIH1975: cancer type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-80: [crRNA sequence]: crRNA sequence: TTTCTGATTTCG A ACTTTCGCGTGTCCT (SEQ ID NO: 317): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGATTTCGAACTTTCGCGTGTCCT (SEQ ID NO: 318): [Target gene information]: Gene ID: 2042: Symbol: EPHA3: Ensembl_ Transcript_ID: ENST00000336596.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 89480460: mut_end: 89480460: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.G766E: mutation info source: CCLE: ref_target(−10 + 10): TCTGATTTCG G ACTTTCGCGT (SEQ ID NO: 319): mut_target(−10 + 10): TCTGATTTCG A ACTTTCGCGT (SEQ ID NO: 320): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-81: [crRNA sequence]: crRNA sequence: TTTCG A ACTTTCGCGTGTCCTGGAGGAT (SEQ ID NO: 321): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAACTTTCGCGTGTCCTGGAGGAT (SEQ ID NO: 322): [Target gene information]: Gene ID: 2042: Symbol: EPHA3: Ensembl_ Transcript_ID: ENST00000336596.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 89480460: mut_end: 89480460: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.G766E: mutation info source: CCLE: ref_target(−10 + 10): TCTGATTTCG G ACTTTCGCGT (SEQ ID NO: 319): mut_target(−10 + 10): TCTGATTTCG A ACTTTCGCGT (SEQ ID NO: 320): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-82: [crRNA sequence]: crRNA sequence: TTTGCACACAAGGTTACTGTTG T TTAAG (SEQ ID NO: 323): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CACACAAGGTTACTGTTGTTTAAG (SEQ ID NO: 324): [Target gene information]: Gene ID: 2044: Symbol: EPHA5: Ensembl_ Transcript_ID: ENST00000273854.3: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 66217192: mut_end: 66217192: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: T: mut_aa: p.I808N: mutation info source: CCLE: ref_target(−10 + 10): GTTACTGTTG A TTAAGATGTT (SEQ ID NO: 325): mut_target(−10 + 10): GTTACTGTTG T TTAAGATGTT (SEQ ID NO: 326): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-83: [crRNA sequence]: crRNA sequence:  T TTAAGATGTTTCTGGCAGCAAGATCTC (SEQ ID NO: 327): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGATGTTTCTGGCAGCAAGATCTC (SEQ ID NO: 328): [Target gene information]: Gene ID: 2044: Symbol: EPHA5: Ensembl_ Transcript_ID: ENST00000273854.3: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 66217192: mut_end: 66217192: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: T: mut_aa: p.I808N: mutation info source: CCLE: ref_target(−10 + 10): GTTACTGTTG A TTAAGATGTT (SEQ ID NO: 325): mut_target(−10 + 10): GTTACTGTTG T TTAAGATGTT (SEQ ID NO: 326): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-84: [crRNA sequence]: crRNA sequence: TTTCATAACAC C GTCACCAAGATCAAGT (SEQ ID NO: 329): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATAACACCGTCACCAAGATCAAGT (SEQ ID NO: 330): [Target gene information]: Gene ID: 2044: Symbol: EPHA5: Ensembl_ Transcript_ID: ENST00000273854.3: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 66467698: mut_end: 66467698: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: C: mut_aa: p.R191G: mutation info source: CCLE: ref_target(−10 + 10): TTCATAACAC G GTCACCAAGA (SEQ ID NO: 331): mut_target(−10 + 10): TTCATAACAC C GTCACCAAGA (SEQ ID NO: 332): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-85: [crRNA sequence]: crRNA sequence: TTTACAGAACTTGATCTTGGTGAC G GTG (SEQ ID NO: 333): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGAACTTGATCTTGGTGACGGTG (SEQ ID NO: 334): [Target gene information]: Gene ID: 2044: Symbol: EPHA5: Ensembl_ Transcript_ID: ENST00000273854.3: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 66467698: mut_end: 66467698: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: C: mut_aa: p.R191G: mutation info source: CCLE: ref_target(−10 + 10): TTCATAACAC G GTCACCAAGA (SEQ ID NO: 331): mut_target(−10 + 10): TTCATAACAC C GTCACCAAGA (SEQ ID NO: 332): [Model Cell line information]: cell: NCIH2126: cancer type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-86: [crRNA sequence]: crRNA sequence: TTT T TTTTTCTGTGTAACCAACTTTCAG (SEQ ID NO: 335): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTCTGTGTAACCAACTTTCAG (SEQ ID NO: 336): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93967904: mut_end: 93967904: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q675K: mutation info source: CCLE: ref_target(−10 + 10): TCTCTCCTTT G TTTTTCTGTG (SEQ ID NO: 337): mut_target(−10 + 10): TCTCTCCTTT T TTTTTCTGTG (SEQ ID NO: 338): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-87: [crRNA sequence]: crRNA sequence: TT T TTTTTCTGTGTAACCAACTTTCAGG (SEQ ID NO: 339): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTCTGTGTAACCAACTTTCAGG (SEQ ID NO: 340): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93967904: mut_end: 93967904: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q675K: mutation info source: CCLE: ref_target(−10 + 10): TCTCTCCTTT G TTTTTCTGTG (SEQ ID NO: 337): mut_target(−10 + 10): TCTCTCCTTT T TTTTTCTGTG (SEQ ID NO: 338): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-88: [crRNA sequence]: crRNA sequence: T T TTTTTCTGTGTAACCAACTTTCAGGG (SEQ ID NO: 341): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCTGTGTAACCAACTTTCAGGG (SEQ ID NO: 342): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93967904: mut_end: 93967904: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q675K: mutation info source: CCLE: ref_target(−10 + 10): TCTCTCCTTT G TTTTTCTGTG (SEQ ID NO: 337): mut_target(−10 + 10): TCTCTCCTTT T TTTTTCTGTG (SEQ ID NO: 338): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-89: [crRNA sequence]: crRNA sequence:  T TTTTTCTGTGTAACCAACTTTCAGGGT (SEQ ID NO: 343): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCTGTGTAACCAACTTTCAGGGT (SEQ ID NO: 344): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93967904: mut_end: 93967904: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q675K: mutation info source: CCLE: ref_target(−10 + 10): TCTCTCCTTT G TTTTTCTGTG (SEQ ID NO: 337): mut_target(−10 + 10): TCTCTCCTTT T TTTTTCTGTG (SEQ ID NO: 338): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-90: [crRNA sequence]: crRNA sequence: TTTCATCTGCAGCAATGGTGT T TATTTT (SEQ ID NO: 345): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATCTGCAGCAATGGTGTTTATTTT (SEQ ID NO: 346): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94120612: mut_end: 94120612: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.D147N: mutation info source: CCLE: ref_target(−10 + 10): GCAATGGTGT C TATTTTTACA (SEQ ID NO: 347): mut_target(−10 + 10): GCAATGGTGT T TATTTTTACA (SEQ ID NO: 348): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-91: [crRNA sequence]: crRNA sequence: T T TATTTTTACATAGAGGTTTTCTCTTA (SEQ ID NO: 349): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTACATAGAGGTTTTCTCTTA (SEQ ID NO: 350): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94120612: mut_end: 94120612: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.D147N: mutation info source: CCLE: ref_target(−10 + 10): GCAATGGTGT C TATTTTTACA (SEQ ID NO: 347): mut_target(−10 + 10): GCAATGGTGT T TATTTTTACA (SEQ ID NO: 348): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-92: [crRNA sequence]: crRNA sequence: TTTCTTTTGATGACCAACCA T TGTGATC (SEQ ID NO: 351): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTGATGACCAACCATTGTGATC (SEQ ID NO: 352): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93953233: mut_end: 93953233: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L970M: mutation info source: CCLE: ref_target(−10 + 10): TGACCAACCA G TGTGATCCCT (SEQ ID NO: 353): mut_target(−10 + 10): TGACCAACCA T TGTGATCCCT (SEQ ID NO: 354): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-93: [crRNA sequence]: crRNA sequence: TTTGATGACCAACCA T TGTGATCCCTAA (SEQ ID NO: 355): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATGACCAACCATTGTGATCCCTAA (SEQ ID NO: 356): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93953233: mut_end: 93953233: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L970M: mutation info source: CCLE: ref_target(−10 + 10): TGACCAACCA G TGTGATCCCT (SEQ ID NO: 353): mut_target(−10 + 10): TGACCAACCA T TGTGATCCCT (SEQ ID NO: 354): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-94: [crRNA sequence]: crRNA sequence: TTTTCTTTTGATGACCAACCA T TGTGAT (SEQ ID NO: 357): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTTGATGACCAACCATTGTGAT (SEQ ID NO: 358): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93953233: mut_end: 93953233: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L970M: mutation info source: CCLE: ref_target(−10 + 10): TGACCAACCA G TGTGATCCCT (SEQ ID NO: 353): mut_target(−10 + 10): TGACCAACCA T TGTGATCCCT (SEQ ID NO: 354): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 18: indel length: 0:: CRISPR gRNA ID: GF-CCELg12-95: [crRNA sequence]: crRNA sequence: TTTTGATGACCAACCA T TGTGATCCCTA (SEQ ID NO: 359): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GATGACCAACCATTGTGATCCCTA (SEQ ID NO: 360): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93953233: mut_end: 93953233: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L970M: mutation info source: CCLE: ref_target(−10 + 10): TGACCAACCA G TGTGATCCCT (SEQ ID NO: 353): mut_target(−10 + 10): TGACCAACCA T TGTGATCCCT (SEQ ID NO: 354): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-96: [crRNA sequence]: crRNA sequence: TTTAGGGATCACA A TGGTTGGTCATCAA (SEQ ID NO: 361): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGGATCACAATGGTTGGTCATCAA (SEQ ID NO: 362): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93953233: mut_end: 93953233: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L970M: mutation info source: CCLE: ref_target(−10 + 10): TGACCAACCA G TGTGATCCCT (SEQ ID NO: 353): mut_target(−10 + 10): TGACCAACCA T TGTGATCCCT (SEQ ID NO: 354): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-97: [crRNA sequence]: crRNA sequence:  T TTGGGAGCAGGGCGAATGTGTTCCCTG (SEQ ID NO: 363): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGAGCAGGGCGAATGTGTTCCCTG (SEQ ID NO: 364): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94066641: mut_end: 94066641: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.S373I: mutation info source: CCLE: ref_target(−10 + 10): CTGCTCCCAA C TGCACCGCTT (SEQ ID NO: 365): mut_target(−10 + 10): CTGCTCCCAA A TGCACCGCTT (SEQ ID NO: 366): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-98: [crRNA sequence]: crRNA sequence: TTTGTTGTGCTTTAGAATCCAGCAGTA A (SEQ ID NO: 367): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGTGCTTTAGAATCCAGCAGTAA (SEQ ID NO: 368): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-99: [crRNA sequence]: crRNA sequence: TTTAGAATCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 371): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAATCCAGCAGTAATACTGAAAAA (SEQ ID NO: 372): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type:LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-100: [crRNA sequence]: crRNA sequence: TTTCTTTTTCAGTA T TACTGCTGGATTC (SEQ ID NO: 373): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTCAGTATTACTGCTGGATTC (SEQ ID NO: 374): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-101: [crRNA sequence]: crRNA sequence: TTTCAGTA T TACTGCTGGATTCTAAAGC (SEQ ID NO: 375): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGTATTACTGCTGGATTCTAAAGC (SEQ ID NO: 376): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-102: [crRNA sequence]: crRNA sequence: TTTTTCAGTA T TACTGCTGGATTCTAAA (SEQ ID NO: 377): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCAGTATTACTGCTGGATTCTAAA (SEQ ID NO: 378): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-103: [crRNA sequence]: crRNA sequence: TTTTCAGTA T TACTGCTGGATTCTAAAG (SEQ ID NO: 379): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGTATTACTGCTGGATTCTAAAG (SEQ ID NO: 380): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 94124483: mut_end: 94124483: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L34L: mutation info source: CCLE: ref_target(−10 + 10): TCCAGCAGTA G TACTGAAAAA (SEQ ID NO: 369): mut_target(−10 + 10): TCCAGCAGTA A TACTGAAAAA (SEQ ID NO: 370): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-104: [crRNA sequence]: crRNA sequence: TTTCAGACTATT TG GTTTCGAATCATTT (SEQ ID NO: 381): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGACTATTTGGTTTCGAATCATTT (SEQ ID NO: 382): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93956546: mut_end: 93956546: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: G: mut_seq: -: mut_aa: p.P897fs: mutation info source: CCLE: ref_target(−10 + 10): CAGACTATTT G GGTTTCGAAT (SEQ ID NO: 383): mut_target(−10 + 10): CAGACTATTT-GGTTTCGAAT (SEQ ID NO: 384): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 10: indel length: 1: CRISPR gRNA ID: GF-CCELg12-105: [crRNA sequence]: crRNA sequence: TT TG GTTTCGAATCATTTTGTCTAGAAT (SEQ ID NO: 385): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTTCGAATCATTTTGTCTAGAAT (SEQ ID NO: 386): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93956546: mut_end: 93956546: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: G: mut_seq: -: mut_aa: p.P897fs: mutation info source: CCLE: ref_target(−10 + 10): CAGACTATTT G GGTTTCGAAT (SEQ ID NO: 383): mut_target(−10 + 10): CAGACTATTT-GGTTTCGAAT (SEQ ID NO: 384): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 0: indel length: 1: CRISPR gRNA ID: GF-CCELg12-106: [crRNA sequence]: crRNA sequence: TTTTCAGACTATT TG GTTTCGAATCATT (SEQ ID NO: 387): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGACTATTTGGTTTCGAATCATT (SEQ ID NO: 388): [Target gene information]: Gene ID: 2045: Symbol: EPHA7: Ensembl_ Transcript_ID: ENST00000369303.4: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 93956546: mut_end: 93956546: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: G: mut_seq: -: mut_aa: p.P897fs: mutation info source: CCLE: ref_target(−10 + 10): CAGACTATTT G GGTTTCGAAT (SEQ ID NO: 383): mut_target(−10 + 10): CAGACTATTT-GGTTTCGAAT (SEQ ID NO: 384): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 11: indel length: 1: CRISPR gRNA ID: GF-CCELg12-107: [crRNA sequence]: crRNA sequence: TTTGCAGCCTGT A AGGGAACAGGCTCTG (SEQ ID NO: 389): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGCCTGTAAGGGAACAGGCTCTG (SEQ ID NO: 390): [Target gene information]: Gene ID: 2065: Symbol: ERBB3: Ensembl_ Transcript_ID: ENST00000267101.3: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 56482537: mut_end: 56482537: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E332K: mutation info source: CCLE: ref_target(−10 + 10): TGCAGCCTGT G AGGGAACAGG (SEQ ID NO: 391): mut_target(−10 + 10): TGCAGCCTGT A AGGGAACAGG (SEQ ID NO: 392): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-108: [crRNA sequence] crRNA sequence: TTTCCAG T TGGAACCCAATCCCCACACC (SEQ ID NO: 393): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGTTGGAACCCAATCCCCACACC (SEQ ID NO: 394): [Target gene information]: Gene ID: 2065: Symbol: ERBB3: Ensembl_ Transcript_ID: ENST00000267101.3: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 56481886: mut_end: 56481886: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.L272L: mutation info source: CCLE: ref_target(−10 + 10): AACTTTCCAG C TGGAACCCAA (SEQ ID NO: 395): mut_target(−10 + 10): AACTTTCCAG T TGGAACCCAA (SEQ ID NO: 396): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-109: [crRNA sequence] crRNA sequence: TTT G GGATTGGACCGGAATAAGTTAATA (SEQ ID NO: 397): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGATTGGACCGGAATAAGTTAATA (SEQ ID NO: 398): [Target gene information]: Gene ID: 2073: Symbol: ERCC5: Ensembl_ Transcript_ID: ENST00000355739.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 103520461: mut_end: 103520461: mut_class: Splice_Site: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): GTTACTCTTT A GGATTGGACC (SEQ ID NO: 399): mut_target(−10 + 10): GTTACTCTTT G GGATTGGACC (SEQ ID NO: 400): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-110: [crRNA sequence]: crRNA sequence: TTTATTAACTTATTCCGGTCCAATCC C A (SEQ ID NO: 401): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAACTTATTCCGGTCCAATCCCA (SEQ ID NO: 402): [Target gene information]: Gene ID: 2073: Symbol: ERCC5: Ensembl_ Transcript_ID: ENST00000355739.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 103520461: mut_end: 103520461: mut_class: Splice_Site: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): GTTACTCTTT A GGATTGGACC (SEQ ID NO: 399): mut_target(−10 + 10): GTTACTCTTT G GGATTGGACC (SEQ ID NO: 400): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-111: [crRNA sequence]: crRNA sequence: TTTCCCCCCACTCAA T AGCGTGTCTCCG (SEQ ID NO: 403): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCCCCACTCAATAGCGTGTCTCCG (SEQ ID NO: 404): [Target gene information]: Gene ID: 2099: Symbol: ESR1: Ensembl_ Transcript_ID: ENST00000206249.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 152129350: mut_end: 152129350: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.N101N: mutation info source: CCLE: ref_target(−10 + 10): CCCCACTCAA C AGCGTGTCTC (SEQ ID NO: 405): mut_target(−10 + 10): CCCCACTCAA T AGCGTGTCTC (SEQ ID NO: 406): [Model Cell line information]: cell: NCIH1650: cancer_type: LUNG PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-112: [crRNA sequence] crRNA sequence: TTTGTACCAGACTATCAGGCTGAAAGT A (SEQ ID NO: 407): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TACCAGACTATCAGGCTGAAAGTA (SEQ ID NO: 408): [Target gene information]: Gene ID: 2115: Symbol: ETV1: Ensembl_ Transcript_ID: ENST00000430479.1: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 14017052: mut_end: 14017052: mut_class: Splice_Site: mut_type: SNP: ref_seq: A: mut_seq: T: mut_aa: p.L79M: mutation info source: CCLE: ref_target(−10 + 10): CTATACTTAC A ACTTTCAGCC (SEQ ID NO: 409): mut_target(−10 + 10): CTATACTTAC T ACTTTCAGCC (SEQ ID NO: 410): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-113: [crRNA sequence]: crRNA sequence: TTTCAAGGCTTCATACATCTTCC C TAGG (SEQ ID NO: 411): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGGCTTCATACATCTTCCCTAGG (SEQ ID NO: 412): [Target gene information]: Gene ID: 2176: Symbol: FANCC: Ensembl_ Transcript_ID: ENST00000289081.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 98011438: mut_end: 98011438: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.R46G: mutation info source: CCLE: ref_target(−10 + 10): TACATCTTCC T TAGGAACTCC (SEQ ID NO: 413): mut_target(−10 + 10): TACATCTTCC C TAGGAACTCC (SEQ ID NO: 414): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-114: [crRNA sequence]: crRNA sequence: TTTCTGCATTTGCTTCAATATC A CGACT (SEQ ID NO: 415): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGCATTTGCTTCAATATCACGACT (SEQ ID NO: 416): [Target gene information]: Gene ID: 2195: Symbol: FAT1: Ensembl_ Transcript_ID: ENST00000441802.2: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 187532622: mut_end: 187532622: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.R3257R: mutation info source: CCLE: ref_target(−10 + 10): CTTCAATATC C CGACTTGCTG (SEQ ID NO: 417): mut_target(−10 + 10): CTTCAATATC A CGACTTGCTG (SEQ ID NO: 418): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-115: [crRNA sequence]: crRNA sequence: TTTGCTTCAATATC A CGACTTGCTGCAT (SEQ ID NO: 419): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTCAATATCACGACTTGCTGCAT (SEQ ID NO: 420): [Target gene information]: Gene ID: 2195: Symbol: FAT1: Ensembl_ Transcript_ID: ENST00000441802.2: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 187532622: mut_end: 187532622: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.R3257R: mutation info source: CCLE: ref_target(−10 + 10): CTTCAATATC C CGACTTGCTG (SEQ ID NO: 417): mut_target(−10 + 10): CTTCAATATC A CGACTTGCTG (SEQ ID NO: 418): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-116: [crRNA sequence]: crRNA sequence: TTTGTGTTTGAGGCTCTGATCCG T CACA (SEQ ID NO: 421): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTTTGAGGCTCTGATCCGTCACA (SEQ ID NO: 422): [Target gene information]: Gene ID: 55294: Symbol: FBW7: Ensembl_ Transcript_ID: ENST00000281708.4: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 153244138: mut_end: 153244138: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.W673*: mutation info source: CCLE: ref_target(−10 + 10): CTCTGATCCG C CACACAACTC (SEQ ID NO: 423): mut_target(−10 + 10): CTCTGATCCG T CACACAACTC (SEQ ID NO: 424): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-117: [crRNA sequence]: crRNA sequence: TTTGAGGCTCTGATCCG T CACACAACTC (SEQ ID NO: 425): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGCTCTGATCCGTCACACAACTC (SEQ ID NO: 426): [Target gene information]: Gene ID: 55294: Symbol: FBW7: Ensembl_ Transcript_ID: ENST00000281708.4: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 153244138: mut_end: 153244138: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.W673*: mutation info source: CCLE: ref_target(−10 + 10): CTCTGATCCG C CACACAACTC (SEQ ID NO: 423): mut_target(−10 + 10): CTCTGATCCG T CACACAACTC (SEQ ID NO: 424): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-118: [crRNA sequence]: crRNA sequence: TTTGCTGCCTTGTCATAC A CTGAAGAAA (SEQ ID NO: 427): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGCCTTGTCATACACTGAAGAAA (SEQ ID NO: 428): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-119: [crRNA sequence]: crRNA sequence: TTTCTTCAG T GTATGACAAGGCAGCAAA (SEQ ID NO: 431): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCAGTGTATGACAAGGCAGCAAA (SEQ ID NO: 432): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-120: [crRNA sequence]: crRNA sequence: TTTATTTTTTCTTCAG T GTATGACAAGG (SEQ ID NO: 433): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTCTTCAGTGTATGACAAGG (SEQ ID NO: 434): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429):  mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 13: indel length: 0:: CRISPR gRNA ID: GF-CCELg12-121: [crRNA sequence]: crRNA sequence: TTTTTATTTTTTCTTCAG T GTATGACAA (SEQ ID NO: 435): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TATTTTTTCTTCAGTGTATGACAA (SEQ ID NO: 436): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-122: [crRNA sequence]: crRNA sequence: TTTTATTTTTTCTTCAG T GTATGACAAG (SEQ ID NO: 437): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTTTTTCTTCAGTGTATGACAAG (SEQ ID NO: 438): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-123: [crRNA sequence]: crRNA sequence: TTTTTTCTTCAG T GTATGACAAGGCAGC (SEQ ID NO: 439): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCTTCAGTGTATGACAAGGCAGC (SEQ ID NO: 440): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430: [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-124: [crRNA sequence]: crRNA sequence: TTTTTCTTCAG T GTATGACAAGGCAGCA (SEQ ID NO: 441): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTCAGTGTATGACAAGGCAGCA (SEQ ID NO: 442): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-125: [crRNA sequence]: crRNA sequence: TTTTCTTCAG T GTATGACAAGGCAGCAA (SEQ ID NO: 443): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTCAGTGTATGACAAGGCAGCAA (SEQ ID NO: 444): [Target gene information]: Gene ID: 2271: Symbol: FH: Ensembl_ Transcript_ID: ENST00000366560.3: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 241661270: mut_end: 241661270: mut_class: Splice_Site: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.G464V: mutation info source: CCLE: ref_target(−10 + 10): CTTGTCATAC C CTGAAGAAAA (SEQ ID NO: 429): mut_target(−10 + 10): CTTGTCATAC A CTGAAGAAAA (SEQ ID NO: 430): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-126: [crRNA sequence]: crRNA sequence: TTTATCTTC T TGAAAGCCGGAGCTCGCA (SEQ ID NO: 445): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTCTTGAAAGCCGGAGCTCGCA (SEQ ID NO: 446): [Target gene information]: Gene ID: 2321: Symbol: FLT1: Ensembl_ Transcript_ID: ENST00000282397.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28896964: mut_end: 28896964: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q972Q: mutation info source: CCLE: ref_target(−10 + 10): TTTTATCTTC C TGAAAGCCGG (SEQ ID NO: 447): mut_target(−10 + 10): TTTTATCTTC T TGAAAGCCGG (SEQ ID NO: 448): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-127: [crRNA sequence]: crRNA sequence: TTTTATCTTC T TGAAAGCCGGAGCTCGC (SEQ ID NO: 449): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATCTTCTTGAAAGCCGGAGCTCGC (SEQ ID NO: 450): [Target gene information]: Gene ID: 2321: Symbol: FLT1: Ensembl_ Transcript_ID: ENST00000282397.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28896964: mut_end: 28896964: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q972Q: mutation info source: CCLE: ref_target(−10 + 10): TTTTATCTTC C TGAAAGCCGG (SEQ ID NO: 447): mut_target(−10 + 10): TTTTATCTTC T TGAAAGCCGG (SEQ ID NO: 448): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-128: [crRNA sequence] crRNA sequence: TTTCA A GAAGATAAAAGTCTGAGTGATG (SEQ ID NO: 451): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGAAGATAAAAGTCTGAGTGATG (SEQ ID NO: 452): [Target gene information]: Gene ID: 2321: Symbol: FLT1: Ensembl_ Transcript_ID: ENST00000282397.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28896964: mut_end: 28896964: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q972Q: mutation info source: CCLE: ref_target(−10 + 10): TTTTATCTTC C TGAAAGCCGG (SEQ ID NO: 447): mut_target(−10 + 10): TTTTATCTTC T TGAAAGCCGG (SEQ ID NO: 448): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-129: [crRNA sequence] crRNA sequence: TTTGCGAGCTCCGGCTTTCA A GAAGATA (SEQ ID NO: 453): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGAGCTCCGGCTTTCAAGAAGATA (SEQ ID NO: 454): [Target gene information]: Gene ID: 2321: Symbol: FLT1: Ensembl_ Transcript_ID: ENST00000282397.4: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28896964: mut_end: 28896964: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q972Q: mutation info source: CCLE: ref_target(−10 + 10): TTTTATCTTC C TGAAAGCCGG (SEQ ID NO: 447): mut_target(−10 + 10): TTTTATCTTC T TGAAAGCCGG (SEQ ID NO: 448): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-130: [crRNA sequence] crRNA sequence:  T TTTCTTGCCACTGATGATACAAAAGCA (SEQ ID NO: 455): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGCCACTGATGATACAAAAGCA (SEQ ID NO: 456): [Target gene information]: Gene ID: 2322: Symbol: FLT3: Ensembl_ Transcript_ID: ENST00000241453.7: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28623588: mut_end: 28623588: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.N323K: mutation info source: CCLE: ref_target(−10 + 10): ATCCGGTGTC G TTTCTTGCCA (SEQ ID NO: 457): mut_target(−10 + 10): ATCCGGTGTC T TTTCTTGCCA (SEQ ID NO: 458): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-131: [crRNA sequence]: crRNA sequence: TTTGTATCATCAGTGGCAAGAAA A GACA (SEQ ID NO: 459): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TATCATCAGTGGCAAGAAAAGACA (SEQ ID NO: 460): [Target gene information]: Gene ID: 2322: Symbol: FLT3: Ensembl_ Transcript_ID: ENST00000241453.7: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28623588: mut_end: 28623588: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.N323K: mutation info source: CCLE: ref_target(−10 + 10): ATCCGGTGTC G TTTCTTGCCA (SEQ ID NO: 457): mut_target(−10 + 10): ATCCGGTGTC T TTTCTTGCCA (SEQ ID NO: 458): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-132: [crRNA sequence]: crRNA sequence: TTTTGTATCATCAGTGGCAAGAAA A GAC (SEQ ID NO: 461): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTATCATCAGTGGCAAGAAAAGAC (SEQ ID NO: 462): [Target gene information]: Gene ID: 2322: Symbol: FLT3: Ensembl_ Transcript_ID: ENST00000241453.7: GRCh: 37: Chr: 13: [Target cancer mutation information]: mut_start: 28623588: mut_end: 28623588: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.N323K: mutation info source: CCLE: ref_target(−10 + 10): ATCCGGTGTC G TTTCTTGCCA (SEQ ID NO: 457): mut_target(−10 + 10): ATCCGGTGTC T TTTCTTGCCA (SEQ ID NO: 458): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_ dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-133: [crRNA sequence]: crRNA sequence:  T TTTCCATCCTTGTACCTGGCCAGGGAA (SEQ ID NO: 463): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCATCCTTGTACCTGGCCAGGGAA (SEQ ID NO: 464): [Target gene information]: Gene ID: 2324: Symbol: FLT4: Ensembl_ Transcript_ID: ENST00000261937.6: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 180053250: mut_end: 180053250: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.K373K: mutation info source: CCLE: ref_target(−10 + 10): CGGACAGTGC C TTTCCATCCT (SEQ ID NO: 465): mut_target(−10 + 10): CGGACAGTGC T TTTCCATCCT (SEQ ID NO: 466): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-134: [crRNA sequence]: crRNA sequence:  T TTCACACGAATGTTTGCTTACTTCCGA (SEQ ID NO: 467): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACACGAATGTTTGCTTACTTCCGA (SEQ ID NO: 468): [Target gene information]: Gene ID: 27086: Symbol: FOXP1: Ensembl_ Transcript_ID: ENST00000318789.4: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 71026140: mut_end: 71026140: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.W494C: mutation info source: CCLE: ref_target(−10 + 10): TTCGTGTGAA C CAGTTATAGA (SEQ ID NO: 469): mut_target(−10 + 10): TTCGTGTGAA A CAGTTATAGA (SEQ ID NO: 470): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-135: [crRNA sequence]: crRNA sequence: TTTCAAC G CTGACCTGAGGTTTCAGAGC (SEQ ID NO: 471): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AACGCTGACCTGAGGTTTCAGAGC (SEQ ID NO: 472): [Target gene information]: Gene ID: 440093: Symbol: H3F3C: Ensembl_ Transcript_ID: ENST00000340398.3: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 31944863: mut_end: 31944863: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut aa: p.T80A: mutation info source: CCLE: ref_target(−10 + 10): CTCAGGTCAG T GTTGAAATCC (SEQ ID NO: 473): mut_target(−10 + 10): CTCAGGTCAG C GTTGAAATCC (SEQ ID NO: 474): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_ dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-136: [crRNA sequence]: crRNA sequence: TT T CGGCAATAATTATCATCAAAGCCCT (SEQ ID NO: 475): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGCAATAATTATCATCAAAGCCCT (SEQ ID NO: 476): [Target gene information]: Gene ID: 3082: Symbol: HGF: Ensembl_ Transcript_ID: ENST00000222390.5: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 81372751: mut_end: 81372751: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R261R: mutation info source: CCLE: ref_target(−10 + 10): CATCGGGATT G CGGCAATAAT (SEQ ID NO: 477): mut_target(−10 + 10): CATCGGGATT T CGGCAATAAT (SEQ ID NO: 478): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-137: [crRNA sequence]: crRNA sequence: TTTGATGATAATTATTGCCG A AATCCCG (SEQ ID NO: 479): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATGATAATTATTGCCGAAATCCCG (SEQ ID NO: 480): [Target gene information]: Gene ID: 3082: Symbol: HGF: Ensembl_ Transcript_ID: ENST00000222390.5: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 81372751: mut_end: 81372751: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R261R: mutation info source: CCLE: ref_target(−10 + 10): CATCGGGATT G CGGCAATAAT (SEQ ID NO: 477): mut_target(−10 + 10): CATCGGGATT T CGGCAATAAT (SEQ ID NO: 478): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-138: [crRNA sequence]: crRNA sequence: TTTGAA T ACACCAACCTCTGTGCTATTC (SEQ ID NO: 481): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATACACCAACCTCTGTGCTATTC (SEQ ID NO: 482): [Target gene information]: Gene ID: 8356: Symbol: HIST1H3J: Ensembl_ Transcript_ID: ENST00000359303.2: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 27858252: mut_end: 27858252: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.D107Y: mutation info source: CCLE: ref_target(−10 + 10): AGGTTGGTGT C TTCAAAGAGA (SEQ ID NO: 483): mut_target(−10 + 10): AGGTTGGTGT A TTCAAAGAGA (SEQ ID NO: 484): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-139: [crRNA sequence]: crRNA sequence: TTTGTAGTCCCAAGGCTA C GCAGGAGGT (SEQ ID NO: 485): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAGTCCCAAGGCTACGCAGGAGGT (SEQ ID NO: 486): [Target gene information]: Gene ID: 3575: Symbol: IL7R: Ensembl_ Transcript_ID: ENST00000303115.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 35876425: mut_end: 35876425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.L406R: mutation info source: CCLE: ref_target(−10 + 10): GACCTCCTGC T TAGCCTTGGG (SEQ ID NO: 487): mut_target(−10 + 10): GACCTCCTGC G TAGCCTTGGG (SEQ ID NO: 488): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-140:  [crRNA sequence] crRNA sequence: TTTGTTCCTGAAAATG C GAAATATCTGA (SEQ ID NO: 489): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCCTGAAAATGCGAAATATCTGA (SEQ ID NO: 490): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: CHr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-141: [crRNA sequence]: crRNA sequence: TTTAAATCATTTTGTTCCTGAAAATG C G (SEQ ID NO: 493): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATCATTTTGTTCCTGAAAATGCG (SEQ ID NO: 494): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-142: [crRNA sequence]: crRNA sequence: TTTTAAATCATTTTGTTCCTGAAAATG C (SEQ ID NO: 495): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAATCATTTTGTTCCTGAAAATGC (SEQ ID NO: 496): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-143: [crRNA sequence]: crRNA sequence: TTTTGTTCCTGAAAATG C GAAATATCTG (SEQ ID NO: 497): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTCCTGAAAATGCGAAATATCTG (SEQ ID NO: 498): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-144: [crRNA sequence]: crRNA sequence: TTTCCCCTTCAGATATTTC G CATTTTCA (SEQ ID NO: 499): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCCTTCAGATATTTCGCATTTTCA (SEQ ID NO: 500): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-145: [crRNA sequence]: crRNA sequence: TTTC G CATTTTCAGGAACAAAATGATTT (SEQ ID NO: 501): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCATTTTCAGGAACAAAATGATTT (SEQ ID NO: 502): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.57945: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-146: [crRNA sequence]: crRNA sequence: TTTGTTTTTTCCCCTTCAGATATTTC G C (SEQ ID NO: 503): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTCCCCTTCAGATATTTCGC (SEQ ID NO: 504): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-147: [crRNA sequence]: crRNA sequence: TTTTTTCCCCTTCAGATATTTC G CATTT (SEQ ID NO: 505): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCCCCTTCAGATATTTCGCATTT (SEQ ID NO: 506): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-148: [crRNA sequence]: crRNA sequence: TTTTTCCCCTTCAGATATTTC G CATTTT (SEQ ID NO: 507): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCCCTTCAGATATTTCGCATTTT (SEQ ID NO: 508): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-149: [crRNA sequence]: crRNA sequence: TTTTCCCCTTCAGATATTTC G CATTTTC (SEQ ID NO: 509): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCCCTTCAGATATTTCGCATTTTC (SEQ ID NO: 510): [Target gene information]: Gene ID: 8821: Symbol: INPP4B: Ensembl_ Transcript_ID: ENST00000513000.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 143007402: mut_end: 143007402: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.S794S: mutation info source: CCLE: ref_target(−10 + 10): CCTGAAAATG T GAAATATCTG (SEQ ID NO: 491): mut_target(−10 + 10): CCTGAAAATG C GAAATATCTG (SEQ ID NO: 492): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-150: [crRNA sequence]: crRNA sequence: TTTGCT T AGGTAGGAAGTTTGGGCCCAG (SEQ ID NO: 511): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTAGGTAGGAAGTTTGGGCCCAG (SEQ ID NO: 512): [Target gene information]: Gene ID: 3716: Symbol: JAK1: Ensembl_ Transcript_ID: ENST00000342505.4: GRCh: 37: CHr: 1: [Target cancer mutation information]: mut_start: 65339055: mut_end: 65339055: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.Q161*: mutation info source: CCLE: ref_target(−10 + 10): CTTCCTACCT G AGCAAACAGA (SEQ ID NO: 513): mut_target(−10 + 10): CTTCCTACCT A AGCAAACAGA (SEQ ID NO: 514): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-151: [crRNA sequence]: crRNA sequence: TTTCAGATAAAT G AAACCTTCTAGTCTT (SEQ ID NO: 515): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGATAAATGAAACCTTCTAGTCTT (SEQ ID NO: 516): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-152: [crRNA sequence]: crRNA sequence: TTTATTTTTTCAGATAAAT G AAACCTTC (SEQ ID NO: 519): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTCAGATAAATGAAACCTTC (SEQ ID NO: 520): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: CHr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation : mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-153: [crRNA sequence]: crRNA sequence: TTTTATTTTTTCAGATAAAT G AAACCTT (SEQ ID NO: 521): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTTTTTCAGATAAATGAAACCTT (SEQ ID NO: 522): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-154: [crRNA sequence]: crRNA sequence: TTTTTTCAGATAAAT G AAACCTTCTAGT (SEQ ID NO: 523): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCAGATAAATGAAACCTTCTAGT (SEQ ID NO: 524): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-155: [crRNA sequence]: crRNA sequence: TTTTTCAGATAAAT G AAACCTTCTAGTC (SEQ ID NO: 525): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCAGATAAATGAAACCTTCTAGTC (SEQ ID NO: 526): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-156: [crRNA sequence]: crRNA sequence: TTTTCAGATAAAT G AAACCTTCTAGTCT (SEQ ID NO: 527): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGATAAATGAAACCTTCTAGTCT (SEQ ID NO: 528): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation : mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-157: [crRNA sequence] crRNA sequence: TTT C ATTTATCTGAAAAAATAAAATACA (SEQ ID NO: 529): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTTATCTGAAAAAATAAAATACA (SEQ ID NO: 530): [Target gene information]: Gene ID: 3717: Symbol: JAK2: Ensembl_ Transcript_ID: ENST00000381652.3: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 5069931: mut_end: 5069931: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.S507*: mutation info source: CCLE: ref_target(−10 + 10): TCAGATAAAT C AAACCTTCTA (SEQ ID NO: 517): mut_target(−10 + 10): TCAGATAAAT G AAACCTTCTA (SEQ ID NO: 518): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_ dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-158: [crRNA sequence]: crRNA sequence: TTTGACAGCTTCCTCCTCACTGT A TGTG (SEQ ID NO: 531): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACAGCTTCCTCCTCACTGTATGTG (SEQ ID NO: 532): [Target gene information]: Gene ID: 3718: Symbol: JAK3: Ensembl_ Transcript_ID: ENST00000527670.1: GRCh: 37: Chr: 19: [Target cancer mutation information]: mut_start: 17951048: mut_end: 17951048: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.V415V: mutation info source: CCLE: ref_target(−10 + 10): CCTGGACACA G ACAGTGAGGA (SEQ ID NO: 533): mut_target(−10 + 10): CCTGGACACA T ACAGTGAGGA (SEQ ID NO: 534): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-159: [crRNA sequence]: crRNA sequence: TTTCTGGTGTAGTATAATCAGGGGC A CT (SEQ ID NO: 535): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGGTGTAGTATAATCAGGGGCACT (SEQ ID NO: 536): [Target gene information]: Gene ID: 3791: Symbol: KDR: Ensembl_ Transcript_ID: ENST00000263923.4: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 55955567: mut_end: 55955567: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.R1126S: mutation info source: CCLE: ref_target(−10 + 10): AATCAGGGGC C CTCATTCTAG (SEQ ID NO: 537): mut_target(−10 + 10): AATCAGGGGC A CTCATTCTAG (SEQ ID NO: 538): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-160: [crRNA sequence]: crRNA sequence: TTTCGCCCGGCTCGAGGTGCAGGATGC G (SEQ ID NO: 539): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCCCGGCTCGAGGTGCAGGATGCG (SEQ ID NO: 540): [Target gene information]: Gene ID: 3791: Symbol: KDR: Ensembl_ Transcript_ID: ENST00000263923.4: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 55991456: mut_end: 55991456: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: p.Q2R: mutation info source: CCLE: ref_target(−10 + 10): CACCTTGCTC T GCATCCTGCA (SEQ ID NO: 541): mut_target(−10 + 10): CACCTTGCTC C GCATCCTGCA (SEQ ID NO: 542): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-161: [crRNA sequence]: crRNA sequence: TTTGTTA T AGGTAAATGCTTGGCTTTCT (SEQ ID NO: 543): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTATAGGTAAATGCTTGGCTTTCT (SEQ ID NO: 544): [Target gene information]: Gene ID: 3815: Symbol: KIT: Ensembl_ Transcript_ID: ENST00000288135.5: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 55561945: mut_end: 55561945: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R112I: mutation info source: CCLE: ref_target(−10 + 10): GTGTTTGTTA G AGGTAAATGC (SEQ ID NO: 545): mut_target(−10 + 10): GTGTTTGTTA T AGGTAAATGC (SEQ ID NO: 546): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-162: [crRNA sequence]: crRNA sequence: TTTATGTGTTTGTTA T AGGTAAATGCTT (SEQ ID NO: 547): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTGTTTGTTATAGGTAAATGCTT (SEQ ID NO: 548): [Target gene information]: Gene ID: 3815: Symbol: KIT: Ensembl_ Transcript_ID: ENST00000288135.5: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 55561945: mut_end: 55561945: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R112I: mutation info source: CCLE: ref_target(−10 + 10): GTGTTTGTTA G AGGTAAATGC (SEQ ID NO: 545): mut_target(−10 + 10): GTGTTTGTTA T AGGTAAATGC (SEQ ID NO: 546): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-163: [crRNA sequence] crRNA sequence: TTTACCT A TAACAAACACATAAATGGAA (SEQ ID NO: 549): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTATAACAAACACATAAATGGAA (SEQ ID NO: 550): [Target gene information]: Gene ID: 3815: Symbol: KIT: Ensembl_ Transcript_ID: ENST00000288135.5: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 55561945: mut_end: 55561945: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.R112I: mutation info source: CCLE: ref_target(−10 + 10): GTGTTTGTTA G AGGTAAATGC (SEQ ID NO: 545): mut_target(−10 + 10): GTGTTTGTTA T AGGTAAATGC (SEQ ID NO: 546): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-164: [crRNA sequence] crRNA sequence: TT T CTTAGAACTATTGCCATTGGAGAGA (SEQ ID NO: 551): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAGAACTATTGCCATTGGAGAGA (SEQ ID NO: 552): [Target gene information]: Gene ID: 4297: Symbol: KMT2A: Ensembl_ Transcript_ID: ENST00000389506.5: GRCh: 37: CHr: 11: [Target cancer mutation information]: mut_start: 118354982: mut_end: 118354983: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q1391fs: mutation info source: CCLE: ref_target(−10 + 10): TAGTTCTAAG-AAAAATTCCA (SEQ ID NO: 105): mut_target(−10 + 10): TAGTTCTAAG A AAAAATTCCA (SEQ ID NO: 95): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: −1: indel length: 1: CRISPR gRNA ID: GF-CCELg12-165: [crRNA sequence]: crRNA sequence: TTTTT T CTTAGAACTATTGCCATTGGAG (SEQ ID NO: 89): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCTTAGAACTATTGCCATTGGAG (SEQ ID NO: 98): [Target gene information]: Gene ID: 4297: Symbol: KMT2A: Ensembl_ Transcript_ID: ENST00000389506.5: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 118354982: mut_end: 118354983: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q1391fs: mutation info source: CCLE: ref_target(−10 + 10): TAGTTCTAAG-AAAAATTCCA (SEQ ID NO: 105): mut_target(−10 + 10): TAGTTCTAAG A AAAAATTCCA (SEQ ID NO: 95): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: 2: indel length: 1: CRISPR gRNA ID: GF-CCELg12-166: [crRNA sequence]: crRNA sequence: TTTT T CTTAGAACTATTGCCATTGGAGA (SEQ ID NO: 553): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTAGAACTATTGCCATTGGAGA (SEQ ID NO: 554): [Target gene information]: Gene ID: 4297: Symbol: KMT2A: Ensembl_ Transcript_ID: ENST00000389506.5: GRCh: 37: CHr: 11: [Target cancer mutation information]: mut_start: 118354982: mut_end: 118354983: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q1391fs: mutation info source: CCLE: ref_target(−10 + 10): TAGTTCTAAG-AAAAATTCCA (SEQ ID NO: 105): mut_target(−10 + 10): TAGTTCTAAG A AAAAATTCCA (SEQ ID NO: 95): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: 1: indel length: 1: CRISPR gRNA ID: GF-CCELg12-167: [crRNA sequence]: crRNA sequence: TTT T CTTAGAACTATTGCCATTGGAGAG (SEQ ID NO: 555): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTAGAACTATTGCCATTGGAGAG (SEQ ID NO: 556): [Target gene information]: Gene ID: 4297: Symbol: KMT2A: Ensembl_ Transcript_ID: ENST00000389506.5: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 118354982: mut_end: 118354983: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q1391fs: mutation info source: CCLE: ref_target(−10 + 10): TAGTTCTAAG-AAAAATTCCA (SEQ ID NO: 105): mut_target(−10 + 10): TAGTTCTAAG A AAAAATTCCA (SEQ ID NO: 95): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 0: indel length: 1: CRISPR gRNA ID: GF-CCELg12-168: [crRNA sequence]: crRNA sequence: T T TGCTACATTTTCCACTTCTTCCTCAG (SEQ ID NO: 557): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTACATTTTCCACTTCTTCCTCAG (SEQ ID NO: 558): [Target gene information]: Gene ID: 58508: Symbol: KMT2C: Ensembl_ Transcript_ID: ENST00000262189.6: GRCh: 37: Chr: 7: [Target cancer mutation information]: mut_start: 151919709: mut_end: 151919709: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.D1128N: mutation info source: CCLE: ref_target(−10 + 10): AAACCAATGT C TGCTACATTT (SEQ ID NO: 559): mut_target(−10 + 10): AAACCAATGT T TGCTACATTT (SEQ ID NO: 560): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-169: [crRNA sequence] crRNA sequence:  T TTGCTGAGCAGCATACCCCGGTGTGTA (SEQ ID NO: 561): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGAGCAGCATACCCCGGTGTGTA (SEQ ID NO: 562): [Target gene information]: Gene ID: 8085: Symbol: KMT2D: Ensembl_ Transcript_ID: ENST00000301067.7: GRCh: 37: CHr: 12: [Target cancer mutation information]: mut_start: 49446729: mut_end: 49446729: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.V361F: mutation info source: CCLE: ref_target(−10 + 10): TGCTCAGCAA C GGAGCGGATA (SEQ ID NO: 563): mut_target(−10 + 10): TGCTCAGCAA A GGAGCGGATA (SEQ ID NO: 564): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-170: [crRNA sequence] crRNA sequence: TTTATTGAAACATCAGC C AAGACAAGAC (SEQ ID NO: 565): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGAAACATCAGCCAAGACAAGAC (SEQ ID NO: 566): [Target gene information]: Gene ID: 3845: Symbol: KRAS: Ensembl_ Transcript_ID: ENST00000256078.4: GRCh: 37: CHr: 12: [Target cancer mutation information]: mut_start: 25378560: mut_end: 25378560: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.A146A: mutation info source: CCLE: ref_target(−10 + 10): GTCTTGTCTT T GCTGATGTTT (SEQ ID NO: 567): mut_target(−10 + 10): GTCTTGTCTT G GCTGATGTTT (SEQ ID NO: 568): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-171: [crRNA sequence]: crRNA sequence: TTTTATTGAAACATCAGC C AAGACAAGA (SEQ ID NO: 569): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTGAAACATCAGCCAAGACAAGA (SEQ ID NO: 570): [Target gene information]: Gene ID: 3845: Symbol: KRAS: Ensembl_ Transcript_ID: ENST00000256078.4: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 25378560: mut_end: 25378560: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.A146A: mutation info source: CCLE: ref_target(−10 + 10): GTCTTGTCTT T GCTGATGTTT (SEQ ID NO: 567): mut_target(−10 + 10): GTCTTGTCTT G GCTGATGTTT (SEQ ID NO: 568): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-172: [crRNA sequence]: crRNA sequence: TTTAAAAAATGGATGAGC A TTTATTTCA (SEQ ID NO: 571): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAAATGGATGAGCATTTATTTCA (SEQ ID NO: 572): [Target gene information]: Gene ID: 9113: Symbol: LATS1: Ensembl_ Transcript_ID: ENST00000543571.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 149983243: mut_end: 149983243: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.K1005N: mutation info source: CCLE: ref_target(−10 + 10): ATGGATGAGC T TTTATTTCAT (SEQ ID NO: 573): mut_target(−10 + 10): ATGGATGAGC A TTTATTTCAT (SEQ ID NO: 574): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-173: [crRNA sequence] : crRNA sequence: TTTTAAAAAATGGATGAGC A TTTATTTC (SEQ ID NO: 575): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAAAATGGATGAGCATTTATTTC (SEQ ID NO: 576): [Target gene information]: Gene ID: 9113: Symbol: LATS1: Ensembl_ Transcript_ID: ENST00000543571.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 149983243: mut_end: 149983243: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.K1005N: mutation info source: CCLE: ref_target(−10 + 10): ATGGATGAGC T TTTATTTCAT (SEQ ID NO: 573): mut_target(−10 + 10): ATGGATGAGC A TTTATTTCAT (SEQ ID NO: 574): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-174: [crRNA sequence]: crRNA sequence: TTTAAAAAATGGATGAGCTT A TATTTCA (SEQ ID NO: 577): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAAATGGATGAGCTTATATTTCA (SEQ ID NO: 578): [Target gene information]: Gene ID: 9113: Symbol: LATS1: Ensembl_ Transcript_ID: ENST00000543571.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 149983245: mut_end: 149983245: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.K1005*: mutation info source: CCLE: ref_target(−10 + 10): GGATGAGCTT T TATTTCATCA (SEQ ID NO: 579): mut_target(−10 + 10): GGATGAGCTT A TATTTCATCA (SEQ ID NO: 580): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-175: [crRNA sequence]: crRNA sequence: TTTTAAAAAATGGATGAGCTT A TATTTC (SEQ ID NO: 581): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAAAATGGATGAGCTTATATTTC (SEQ ID NO: 582): [Target gene information]: Gene ID: 9113: Symbol: LATS1: Ensembl_ Transcript_ID: ENST00000543571.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 149983245: mut_end: 149983245: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.K1005*: mutation info source: CCLE: ref_target(−10 + 10): GGATGAGCTT T TATTTCATCA (SEQ ID NO: 579): mut_target(−10 + 10): GGATGAGCTT A TATTTCATCA (SEQ ID NO: 580): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-176: [crRNA sequence]: crRNA sequence: TTTGGT T CTGCGGGTAGCCCCTGAACCG (SEQ ID NO: 583): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTCTGCGGGTAGCCCCTGAACCG (SEQ ID NO: 584): [Target gene information]: Gene ID: 26524: Symbol: LATS2: Ensembl_ Transcript_ID: ENST00000382592.4: GRCh: 37: CHr: 13: [Target cancer mutation information]: mut_start: 21620047: mut_end: 21620047: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G40E: mutation info source: CCLE: ref_target(−10 + 10): ACTGTTTGGT C CTGCGGGTAG (SEQ ID NO: 585): mut_target(−10 + 10): ACTGTTTGGT T CTGCGGGTAG (SEQ ID NO: 586): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-177: [crRNA sequence]: crRNA sequence: TTTCTTACCC C GAAGCAGAAGGTGGGAG (SEQ ID NO: 587): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTACCCCGAAGCAGAAGGTGGGAG (SEQ ID NO: 588): [Target gene information]: Gene ID: 5604: Symbol: MAP2K1: Ensembl_ Transcript_ID: ENST00000307102.5: GRCh: 37: CHr: 15: [Target cancer mutation information]: mut_start: 66727451: mut_end: 66727451: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: C: mut_aa: p.Q56P: mutation info source: CCLE: ref_target(−10 + 10): TTTCTTACCC A GAAGCAGAAG (SEQ ID NO: 589): mut_target(−10 + 10): TTTCTTACCC C GAAGCAGAAG (SEQ ID NO: 590): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-178: [crRNA sequence]: crRNA sequence: TTTAGAAATCAGTCCCCA T AATGTGGAT (SEQ ID NO: 591): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAAATCAGTCCCCATAATGTGGAT (SEQ ID NO: 592): [Target gene information]: Gene ID: 4292: Symbol: MLH1: Ensembl_ Transcript_ID: ENST00000231790.2: GRCh: 37: CHr: 3: [Target cancer mutation information]: mut_start: 37061819: mut_end: 37061819: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q301H: mutation info source: CCLE: ref_target(−10 + 10): TCAGTCCCCA G AATGTGGATG (SEQ ID NO: 593): mut_target(−10 + 10): TCAGTCCCCA T AATGTGGATG (SEQ ID NO: 594): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-179: [crRNA sequence]: crRNA sequence: T TT GTAAGACAGTTTTATTTTACTGTTT (SEQ ID NO: 595): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAAGACAGTTTTATTTTACTGTTT (SEQ ID NO: 596): [Target gene information]: Gene ID: 4361: Symbol: MRE11A: Ensembl_ Transcript_ID: ENST00000323929.3: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 94211901: mut_end: 94211902: mut_class: Splice_Site: mut_type: DNP: ref_seq: CT: mut_seq: AA: mut_aa: p.181 182LG > 2F*: mutation info source: CCLE: ref_target(−10 + 10): ACTGTCTTAC CT AAACCATATA (SEQ ID NO: 597): mut_target(−10 + 10): ACTGTCTTAC AA AAACCATATA (SEQ ID NO: 598): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-180: [crRNA sequence]: crRNA sequence: TTT TT GTAAGACAGTTTTATTTTACTGT (SEQ ID NO: 599): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTAAGACAGTTTTATTTTACTGT (SEQ ID NO: 600): [Target gene information]: Gene ID: 4361: Symbol: MRE11A: Ensembl_ Transcript_ID: ENST00000323929.3: GRCh: 37: CHr: 11: [Target cancer mutation information]: mut_start: 94211901: mut_end: 94211902: mut_class: Splice_Site: mut_type: DNP: ref_seq: CT: mut_seq: AA: mut_aa: p.181 182LG > F*: mutation info source: CCLE: ref_target(−10 + 10): ACTGTCTTAC CT AAACCATATA (SEQ ID NO: 597): mut_target(−10 + 10): ACTGTCTTAC AA AAACCATATA (SEQ ID NO: 598): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-181: [crRNA sequence]: crRNA sequence: TT TT GTAAGACAGTTTTATTTTACTGTT (SEQ ID NO: 601): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTAAGACAGTTTTATTTTACTGTT (SEQ ID NO: 602): [Target gene information]: Gene ID: 4361: Symbol: MRE11A: Ensembl_ Transcript_ID: ENST00000323929.3: GRCh: 37: Chr: 11: [Target cancer mutation information]: mut_start: 94211901: mut_end: 94211902: mut_class: Splice_Site: mut_type: DNP: ref_seq: CT: mut_seq: AA: mut_aa: p.181 182LG > F*: mutation info source: CCLE: ref_target(−10 + 10): ACTGTCTTAC CT AAACCATATA (SEQ ID NO: 597): mut_target(−10 + 10): ACTGTCTTAC AA AAACCATATA (SEQ ID NO: 598): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-182: [crRNA sequence]: crRNA sequence: TTTGGCC T GGTGATCTCACAGCCCACCC (SEQ ID NO: 603): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCCTGGTGATCTCACAGCCCACCC (SEQ ID NO: 604): [Target gene information]: Gene ID: 4595: Symbol: MUTYH: Ensembl_ Transcript_ID: ENST00000372098.3: GRCh: 37: CHr: 1: [Target cancer mutation information]: mut_start: 45798247: mut_end: 45798247: mut_class: Splice_Site: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.Q227L: mutation info source: CCLE: ref_target(−10 + 10): TGAGATCACC T GGCCAAAGGC (SEQ ID NO: 605): mut_target(−10 + 10): TGAGATCACC A GGCCAAAGGC (SEQ ID NO: 606): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-183: [crRNA sequence]: crRNA sequence: TTTCTTTCCTGC T GTCCTGACAGATCAA (SEQ ID NO: 607): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCCTGCTGTCCTGACAGATCAA (SEQ ID NO: 608): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983459: mut_end: 90983459: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R215Q: mutation info source: CCLE: ref_target(−10 + 10): TCTTTCCTGC C GTCCTGACAG (SEQ ID NO: 609): mut_target(−10 + 10): TCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 610): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-184: [crRNA sequence]: crRNA sequence: TTTCCTGC T GTCCTGACAGATCAACATT (SEQ ID NO: 611): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGCTGTCCTGACAGATCAACATT (SEQ ID NO: 612): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983459: mut_end: 90983459: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R215Q: mutation info source: CCLE: ref_target(−10 + 10): TCTTTCCTGC C GTCCTGACAG (SEQ ID NO: 609): mut_target(−10 + 10): TCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 610): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-185: [crRNA sequence]: crRNA sequence: TTTGAAGATTTGTTTTCTTTCCTGC T GT (SEQ ID NO: 613): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGATTTGTTTTCTTTCCTGCTGT (SEQ ID NO: 614): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983459: mut_end: 90983459: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R215Q: mutation info source: CCLE: ref_target(−10 + 10): TCTTTCCTGC C GTCCTGACAG (SEQ ID NO: 609): mut_target(−10 + 10): TCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 610): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-186: [crRNA sequence]: crRNA sequence: TTTGTTTTCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 615): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTCTTTCCTGCTGTCCTGACAG (SEQ ID NO: 616): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983459: mut_end: 90983459: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R215Q: mutation info source: CCLE: ref_target(−10 + 10): TCTTTCCTGC C GTCCTGACAG (SEQ ID NO: 609): mut_target(−10 + 10): TCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 610): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-187: [crRNA sequence]: crRNA sequence: TTTTCTTTCCTGC T GTCCTGACAGATCA (SEQ ID NO: 617): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTCCTGCTGTCCTGACAGATCA (SEQ ID NO: 618): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983459: mut_end: 90983459: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R215Q: mutation info source: CCLE: ref_target(−10 + 10): TCTTTCCTGC C GTCCTGACAG (SEQ ID NO: 609): mut_target(−10 + 10): TCTTTCCTGC T GTCCTGACAG (SEQ ID NO: 610): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-188: [crRNA sequence]: crRNA sequence: TTTC G CTTTGAAGATTTGTTTTCTTTCC (SEQ ID NO: 619): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCTTTGAAGATTTGTTTTCTTTCC (SEQ ID NO: 620): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983432: mut_end: 90983432: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.G224A: mutation info source: CCLE: ref_target(−10 + 10): AAATGTTTTC C CTTTGAAGAT (SEQ ID NO: 621): mut_target(−10 + 10): AAATGTTTTC G CTTTGAAGAT (SEQ ID NO: 622): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-189: [crRNA sequence]: crRNA sequence: TTTTC G CTTTGAAGATTTGTTTTCTTTC (SEQ ID NO: 623): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGCTTTGAAGATTTGTTTTCTTTC (SEQ ID NO: 624): [Target gene information]: Gene ID: 4683: Symbol: NBN: Ensembl_ Transcript_ID: ENST00000265433.3: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 90983432: mut_end: 90983432: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.G224A: mutation info source: CCLE: ref_target(−10 + 10): AAATGTTTTC C CTTTGAAGAT (SEQ ID NO: 621): mut_target(−10 + 10): AAATGTTTTC G CTTTGAAGAT (SEQ ID NO: 622): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-190: [crRNA sequence]: crRNA sequence: TTTCCGTGGTGGGCTCAAGCTCAAC A GC (SEQ ID NO: 625): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGTGGTGGGCTCAAGCTCAACAGC (SEQ ID NO: 626): [Target gene information]: Gene ID: 9611: Symbol: NCOR1: Ensembl_ Transcript_ID: ENST00000268712.3: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 16005004: mut_end: 16005004: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A750A: mutation info source: CCLE: ref_target(−10 + 10): CAAGCTCAAC C GCAGGTTCTG (SEQ ID NO: 627): mut_target(−10 + 10): CAAGCTCAAC A GCAGGTTCTG (SEQ ID NO: 628): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_ dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-191: [crRNA sequence]: crRNA sequence: TTTCGAGGAAATACCCCC T TACACCTTG (SEQ ID NO: 629): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGGAAATACCCCCTTACACCTTG (SEQ ID NO: 630): [Target gene information]: Gene ID: 4792: Symbol: NFKBIA: Ensembl_ Transcript_ID: ENST00000216797.5: GRCh: 37: Chr: 14: [Target cancer mutation information]: mut_start: 35872461: mut_end: 35872461: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L148L: mutation info source: CCLE: ref_target(−10 + 10): GCAAGGTGTA G GGGGGTATTT (SEQ ID NO: 631): mut_target(−10 + 10): GCAAGGTGTA A GGGGGTATTT (SEQ ID NO: 632): [Model Cell line information]: cell: CFPAC1: cancer_type: PANCREAS: PAM_ dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-192: [crRNA sequence]: crRNA sequence: TTTGA C GGTCAGAATTGTGAAGTGAACG (SEQ ID NO: 633): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACGGTCAGAATTGTGAAGTGAACG (SEQ ID NO: 634): [Target gene information]: Gene ID: 4854: Symbol: NOTCH3: Ensembl_ Transcript_ID: ENST00000263388.2: GRCh: 37: Chr: 19: [Target cancer mutation information]: mut_start: 15302671: mut_end: 15302671: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E229D: mutation info source: CCLE: ref_target(−10 + 10): AATTCTGACC C TCAAACCCTA (SEQ ID NO: 635): mut_target(−10 + 10): AATTCTGACC G TCAAACCCTA (SEQ ID NO: 636): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-193: [crRNA sequence]: crRNA sequence: T T TCCAGCTGTATCCAGTATGTCCAACA (SEQ ID NO: 637): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGCTGTATCCAGTATGTCCAACA (SEQ ID NO: 638): [Target gene information]: Gene ID: 4893: Symbol: NRAS: Ensembl_ Transcript_ID: ENST00000369535.4: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 115256530: mut_end: 115256530: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q61K: mutation info source: CCLE: ref_target(−10 + 10): TACTCTTCTT G TCCAGCTGTA (SEQ ID NO: 639): mut_target(−10 + 10): TACTCTTCTT T TCCAGCTGTA (SEQ ID NO: 640): [Model Cell line information]: cell: NCIH1299: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-194: [crRNA sequence]: crRNA sequence: TT T TCCAGCTGTATCCAGTATGTCCAAC (SEQ ID NO: 641): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCAGCTGTATCCAGTATGTCCAAC (SEQ ID NO: 642): [Target gene information]: Gene ID: 4893: Symbol: NRAS: Ensembl_ Transcript_ID: ENST00000369535.4: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 115256530: mut_end: 115256530: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q61K: mutation info source: CCLE: ref_target(−10 + 10): TACTCTTCTT G TCCAGCTGTA (SEQ ID NO: 639): mut_target(−10 + 10): TACTCTTCTT T TCCAGCTGTA (SEQ ID NO: 640): [Model Cell line information]: cell: NCIH1299: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-195: [crRNA sequence]: crRNA sequence: T T TCCAGCTGTATCCAGTATGTCCAACA (SEQ ID NO: 637): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGCTGTATCCAGTATGTCCAACA (SEQ ID NO: 638): [Target gene information]: Gene ID: 4893: Symbol: NRAS: Ensembl_ Transcript_ID: ENST00000369535.4: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 115256530: mut_end: 115256530: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q61K: mutation info source: CCLE: ref_target(−10 + 10): TACTCTTCTT G TCCAGCTGTA (SEQ ID NO: 639): mut_target(−10 + 10): TACTCTTCTT T TCCAGCTGTA (SEQ ID NO: 640): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-196: [crRNA sequence]: crRNA sequence: TT T TCCAGCTGTATCCAGTATGTCCAAC (SEQ ID NO: 641): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCAGCTGTATCCAGTATGTCCAAC (SEQ ID NO: 642): [Target gene information]: Gene ID: 4893: Symbol: NRAS: Ensembl_ Transcript_ID: ENST00000369535.4: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 115256530: mut_end: 115256530: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.Q61K: mutation info source: CCLE: ref_target(−10 + 10): TACTCTTCTT G TCCAGCTGTA (SEQ ID NO: 639): mut_target(−10 + 10): TACTCTTCTT T TCCAGCTGTA (SEQ ID NO: 640): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-197: [crRNA sequence]: crRNA sequence: TT T GCTTTTCCTCATCACCTGCTCCAAT (SEQ ID NO: 643): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTTCCTCATCACCTGCTCCAAT (SEQ ID NO: 644): [Target gene information]: Gene ID: 64324: Symbol: NSD1: Ensembl_ Transcript_ID: ENST00000439151.2: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 176637295: mut_end: 176637295: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.R632Q: mutation info source: CCLE: ref_target(−10 + 10): GAGGAAAAGC G AAGTGATTCC (SEQ ID NO: 645): mut_target(−10 + 10): GAGGAAAAGC A AAGTGATTCC (SEQ ID NO: 646): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-198: [crRNA sequence]: crRNA sequence: TTTGGGGTTGCCTT G CACAGTGAATGGA (SEQ ID NO: 647): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGGTTGCCTTGCACAGTGAATGGA (SEQ ID NO: 648): [Target gene information]: Gene ID: 4915: Symbol: NTRK2: Ensembl_ Transcript_ID: ENST00000323115.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 87342637: mut_end: 87342637: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: C: mut_aa: p.K308Q: mutation info source: CCLE: ref_target(−10 + 10): ATTCACTGTG A AAGGCAACCC (SEQ ID NO: 649): mut_target(−10 + 10): ATTCACTGTG C AAGGCAACCC (SEQ ID NO: 650): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-199: [crRNA sequence]: crRNA sequence: TT T CCTTGTGCCTCCAAACTTACAGGTG (SEQ ID NO: 651): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGTGCCTCCAAACTTACAGGTG (SEQ ID NO: 652): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-200: [crRNA sequence]: crRNA sequence: TTTTTTT T CCTTGTGCCTCCAAACTTAC (SEQ ID NO: 655): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTCCTTGTGCCTCCAAACTTAC (SEQ ID NO: 656): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-201: [crRNA sequence]: crRNA sequence: TTTTTT T CCTTGTGCCTCCAAACTTACA (SEQ ID NO: 657): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCCTTGTGCCTCCAAACTTACA (SEQ ID NO: 658): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-202: [crRNA sequence]: crRNA sequence: TTTTT T CCTTGTGCCTCCAAACTTACAG (SEQ ID NO: 659): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCCTTGTGCCTCCAAACTTACAG (SEQ ID NO: 660): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-203: [crRNA sequence]: crRNA sequence: TTTT T CCTTGTGCCTCCAAACTTACAGG (SEQ ID NO: 661): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCTTGTGCCTCCAAACTTACAGG (SEQ ID NO: 662): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-204: [crRNA sequence]: crRNA sequence: TTT T CCTTGTGCCTCCAAACTTACAGGT (SEQ ID NO: 663): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTTGTGCCTCCAAACTTACAGGT (SEQ ID NO: 664): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-205: [crRNA sequence] crRNA sequence: TTTGGAGGCACAAGG A AAAAAAATGACT (SEQ ID NO: 665): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGGCACAAGGAAAAAAAATGACT (SEQ ID NO: 666): [Target gene information]: Gene ID: 79728: Symbol: PALB2: Ensembl_ Transcript_ID: ENST00000261584.4: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 23646988: mut_end: 23646988: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G293G: mutation info source: CCLE: ref_target(−10 + 10): TCATTTTTTT G CCTTGTGCCT (SEQ ID NO: 653): mut_target(−10 + 10): TCATTTTTTT T CCTTGTGCCT (SEQ ID NO: 654): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-206: [crRNA sequence]: crRNA sequence: TTTCTTAAACCTACCTCATTCGAA T ACT (SEQ ID NO: 667): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAAACCTACCTCATTCGAATACT (SEQ ID NO: 668): [Target gene information]: Gene ID: 55193: Symbol: PBRM1: Ensembl_ Transcript_ID: ENST00000296302.7: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 52678730: mut_end: 52678730: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L297I: mutation info source: CCLE: ref_target(−10 + 10): CTCATTCGAA G ACTTGACTTA (SEQ ID NO: 669): mut_target(−10 + 10): CTCATTCGAA T ACTTGACTTA (SEQ ID NO: 670): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-207: [crRNA sequence]: crRNA sequence: TTTTCTTAAACCTACCTCATTCGAA T AC (SEQ ID NO: 671): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTAAACCTACCTCATTCGAATAC (SEQ ID NO: 672): [Target gene information]: Gene ID: 55193: Symbol: PBR1M1: Ensembl_ Transcript_ID: ENST00000296302.7: GRCh: 37: CHr: 3: [Target cancer mutation information]: mut_start: 52678730: mut_end: 52678730: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L297I: mutation info source: CCLE: ref_target(−10 + 10): CTCATTCGAA G ACTTGACTTA (SEQ ID NO: 669): mut_target(−10 + 10): CTCATTCGAA T ACTTGACTTA (SEQ ID NO: 670): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-208: [crRNA sequence]: crRNA sequence: T T TGTATTCTTCTCATCATCACCTTTAT (SEQ ID NO: 673): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TATTCTTCTCATCATCACCTTTAT (SEQ ID NO: 674): [Target gene information]: Gene ID: 55193: Symbol: PBRM11: Ensembl_ Transcript_ID: ENST00000296302.7: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 52620488: mut_end: 52620488: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.D1114N: mutation info source: CCLE: ref_target(−10 + 10): TCTGAGTTGT C TGTATTCTTC (SEQ ID NO: 675): mut_target(−10 + 10): TCTGAGTTGT T TGTATTCTTC (SEQ ID NO: 676): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-209: [crRNA sequence]: crRNA sequence: TTTATCCCTCCAAA C GTTTGTGCATGAC (SEQ ID NO: 677): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCCTCCAAACGTTTGTGCATGAC (SEQ ID NO: 678): [Target gene information]: Gene ID: 5288: Symbol: PIK3C2G: Ensembl_ Transcript_ID: ENST00000266497.5: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 18658378: mut_end: 18658378: mut_class: Silent: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.T1061T: mutation info source: CCLE: ref_target(−10 + 10): ATGCACAAAC A TTTGGAGGGA (SEQ ID NO: 679): mut_target(−10 + 10): ATGCACAAAC G TTTGGAGGGA (SEQ ID NO: 680): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-210: [crRNA sequence]: crRNA sequence: TTTTTATCCCTCCAAA C GTTTGTGCATG (SEQ ID NO: 681): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TATCCCTCCAAACGTTTGTGCATG (SEQ ID NO: 682): [Target gene information]: Gene ID: 5288: Symbol: PIK3C2G: Ensembl_ Transcript_ID: ENST00000266497.5: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 18658378: mut_end: 18658378: mut_class: Silent: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.T1061T: mutation info source: CCLE: ref_target(−10 + 10): ATGCACAAAC A TTTGGAGGGA (SEQ ID NO: 679): mut_target(−10 + 10): ATGCACAAAC G TTTGGAGGGA (SEQ ID NO: 680): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-211: [crRNA sequence]: crRNA sequence: TTTTATCCCTCCAAA C GTTTGTGCATGA (SEQ ID NO: 683): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATCCCTCCAAACGTTTGTGCATGA (SEQ ID NO: 684): [Target gene information]: Gene ID: 5288: Symbol: PIK3C2G: Ensembl_ Transcript_ID: ENST00000266497.5: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 18658378: mut_end: 18658378: mut_class: Silent: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.T1061T: mutation info source: CCLE: ref_target(−10 + 10): ATGCACAAAC A TTTGGAGGGA (SEQ ID NO: 679): mut_target(−10 + 10): ATGCACAAAC G TTTGGAGGGA (SEQ ID NO: 680): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-212: [crRNA sequence]: crRNA sequence: TTTAAGGGTTAC A TTCAACAATGCTTGG (SEQ ID NO: 685): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGGTTACATTCAACAATGCTTGG (SEQ ID NO: 686): [Target gene information]: Gene ID: 5289: Symbol: PIK3C3: Ensembl_ Transcript_ID: ENST00000262039.4: GRCh: 37: CHr: 18: [Target cancer mutation information]: mut_start: 39607512: mut_end: 39607512: mut_class: Splice_Site: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.K530N: mutation info source: CCLE: ref_target(−10 + 10): CATTGTTGAA G GTAACCCTTA (SEQ ID NO: 687): mut_target(−10 + 10): CATTGTTGAA T GTAACCCTTA (SEQ ID NO: 688): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-213: [crRNA sequence]: crRNA sequence: TTTATGTAATTTTATTAAAG A TTTTGCT (SEQ ID NO: 689): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTAATTTTATTAAAGATTTTGCT (SEQ ID NO: 690): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178917478: mut_end: 178917478: mut_class: Splice_Site: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.G118D: mutation info source: CCLE: ref_target(−10 + 10): TTTATTAAAG G TTTTGCTATC (SEQ ID NO: 691): mut_target(−10 + 10): TTTATTAAAG A TTTTGCTATC (SEQ ID NO: 692): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-214: [crRNA sequence]: crRNA sequence: TTTATTAAAG A TTTTGCTATCGGCATGC (SEQ ID NO: 693): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAAAGATTTTGCTATCGGCATGC (SEQ ID NO: 694): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178917478: mut_end: 178917478: mut_class: Splice_Site: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.G118D: mutation info source: CCLE: ref_target(−10 + 10): TTTATTAAAG G TTTTGCTATC (SEQ ID NO: 691): mut_target(−10 + 10): TTTATTAAAG A TTTTGCTATC (SEQ ID NO: 692): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-215: [crRNA sequence]: crRNA sequence: TTTTATTAAAG A TTTTGCTATCGGCATG (SEQ ID NO: 695): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTAAAGATTTTGCTATCGGCATG (SEQ ID NO: 696): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178917478: mut_end: 178917478: mut_class: Splice_Site: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.G118D: mutation info source: CCLE: ref_target(−10 + 10): TTTATTAAAG G TTTTGCTATC (SEQ ID NO: 691): mut_target(−10 + 10): TTTATTAAAG A TTTTGCTATC (SEQ ID NO: 692): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-216: [crRNA sequence]: crRNA sequence: TTTCTCCTGCT T AGTGATTTCAGAGAGA (SEQ ID NO: 697): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCTGCTTAGTGATTTCAGAGAGA (SEQ ID NO: 698): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178936091: mut_end: 178936091: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E545K: mutation info source: CCLE: ref_target(−10 + 10): TGAAATCACT G AGCAGGAGAA (SEQ ID NO: 699): mut_target(−10 + 10): TGAAATCACT A AGCAGGAGAA (SEQ ID NO: 700): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-217: [crRNA sequence]: crRNA sequence: TTTCTACACGAGATCCTCTCTCT A AAAT (SEQ ID NO: 701): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TACACGAGATCCTCTCTCTAAAAT (SEQ ID NO: 702): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178936082: mut_end: 178936082: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E542K: mutation info source: CCLE: ref_target(−10 + 10): TCCTCTCTCT G AAATCACTGA (SEQ ID NO: 703): mut_target(−10 + 10): TCCTCTCTCT A AAATCACTGA (SEQ ID NO: 704): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-218: [crRNA sequence]: crRNA sequence: TTTCTCCTGCTCAGTGATTT T AGAGAGA (SEQ ID NO: 705): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCTGCTCAGTGATTTTAGAGAGA (SEQ ID NO: 706): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178936082: mut_end: 178936082: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E542K: mutation info source: CCLE: ref_target(−10 + 10): TCCTCTCTCT G AAATCACTGA (SEQ ID NO: 703): mut_target(−10 + 10): TCCTCTCTCT A AAATCACTGA (SEQ ID NO: 704): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-219: [crRNA sequence]: crRNA sequence: TT T AGAGAGAGGATCTCGTGTAGAAATT (SEQ ID NO: 707): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGAGAGGATCTCGTGTAGAAATT (SEQ ID NO: 708): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178936082: mut_end: 178936082: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E542K: mutation info source: CCLE: ref_target(−10 + 10): TCCTCTCTCT G AAATCACTGA (SEQ ID NO: 703): mut_target(−10 + 10): TCCTCTCTCT A AAATCACTGA (SEQ ID NO: 704): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-220: [crRNA sequence]: crRNA sequence: TTT T AGAGAGAGGATCTCGTGTAGAAAT (SEQ ID NO: 709): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGAGAGAGGATCTCGTGTAGAAAT (SEQ ID NO: 710): [Target gene information]: Gene ID: 5290: Symbol: PIK3CA: Ensembl_ Transcript_ID: ENST00000263967.3: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 178936082: mut_end: 178936082: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.E542K: mutation info source: CCLE: ref_target(−10 + 10): TCCTCTCTCT G AAATCACTGA (SEQ ID NO: 703): mut_target(−10 + 10): TCCTCTCTCT A AAATCACTGA (SEQ ID NO: 704): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-221: [crRNA sequence]: crRNA sequence: TTTCCG T GAACTTACGTAGAATATATTG (SEQ ID NO: 711): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGTGAACTTACGTAGAATATATTG (SEQ ID NO: 712): [Target gene information]: Gene ID: 5295: Symbol: PIK3R1: Ensembl_ Transcript_ID: ENST00000521381.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 67522714: mut_end: 67522714: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.G71*: mutation info source: CCLE: ref_target(−10 + 10): GGACTTTCCG G GAACTTACGT (SEQ ID NO: 713): mut_target(−10 + 10): GGACTTTCCG T GAACTTACGT (SEQ ID NO: 714): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-222: [crRNA sequence]: crRNA sequence: TTTGAAAGCGTCAGCCAAAACGTG A ACA (SEQ ID NO: 715): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAAGCGTCAGCCAAAACGTGAACA (SEQ ID NO: 716): [Target gene information]: Gene ID: 5295: Symbol: PIK3R1: Ensembl_ Transcript_ID: ENST00000521381.1: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 67575464: mut_end: 67575464: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.V179V: mutation info source: CCLE: ref_target(−10 + 10): TGATCGATGT G CACGTTTTGG (SEQ ID NO: 717): mut_target(−10 + 10): TGATCGATGT T CACGTTTTGG (SEQ ID NO: 718): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-223: [crRNA sequence]: crRNA sequence: TTTCTCATCATAATGGG C CAGGTTTTCA (SEQ ID NO: 719): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCATCATAATGGGCCAGGTTTTCA (SEQ ID NO: 720): [Target gene information]: Gene ID: 8503: Symbol: PIK3R3: Ensembl_ Transcript_ID: ENST00000262741.5: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 46511726: mut_end: 46511726: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: C: mut_aa: p.P351A: mutation info source: CCLE: ref_target(−10 + 10): TCATAATGGG G CAGGTTTTCA (SEQ ID NO: 721): mut_target(−10 + 10): TCATAATGGG C CAGGTTTTCA (SEQ ID NO: 722): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-224: [crRNA sequence]: crRNA sequence: TTTTCTCATCATAATGGG C CAGGTTTTC (SEQ ID NO: 723): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTCATCATAATGGGCCAGGTTTTC (SEQ ID NO: 724): [Target gene information]: Gene ID: 8503: Symbol: PIK3R3: Ensembl_ Transcript_ID: ENST00000262741.5: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 46511726: mut_end: 46511726: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: C: mut_aa: p.P351A: mutation info source: CCLE: ref_target(−10 + 10): TCATAATGGG G CAGGTTTTCA (SEQ ID NO: 721): mut_target(−10 + 10): TCATAATGGG C CAGGTTTTCA (SEQ ID NO: 722): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_ dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-225: [crRNA sequence]: crRNA sequence: TTTATCAATGAGGAAGATGAAAACCTG G (SEQ ID NO: 725): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCAATGAGGAAGATGAAAACCTGG (SEQ ID NO: 726): [Target gene information]: Gene ID: 8503: Symbol: PIK3R3: Ensembl_ Transcript_ID: ENST00000262741.5: GRCh: 37: Chr: 1: [Target cancer mutation information]: mut_start: 46511726: mut_end: 46511726: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: C: mut_aa: p.P351A: mutation info source: CCLE: ref_target(−10 + 10): TCATAATGGG G CAGGTTTTCA (SEQ ID NO: 721): mut_target(−10 + 10): TCATAATGGG C CAGGTTTTCA (SEQ ID NO: 722): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-226: [crRNA sequence]: crRNA sequence: TTTGCAAAGC T ACCCTGAAAGGAAACAA (SEQ ID NO: 727): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAAAGCTACCCTGAAAGGAAACAA (SEQ ID NO: 728): [Target gene information]: Gene ID: 10769: Symbol: PLK2: Ensembl_ Transcript_ID: ENST00000274289.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 57754916: mut_end: 57754916: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G92S: mutation info source: CCLE: ref_target(−10 + 10): TTTGCAAAGC C ACCCTGAAAG (SEQ ID NO: 729): mut_target(−10 + 10): TTTGCAAAGC T ACCCTGAAAG (SEQ ID NO: 730): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-227: [crRNA sequence]: crRNA sequence: TTTTGCAAAGC T ACCCTGAAAGGAAACA (SEQ ID NO: 731): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCAAAGCTACCCTGAAAGGAAACA (SEQ ID NO: 732): [Target gene information]: Gene ID: 10769: Symbol: PLK2: Ensembl_ Transcript_ID: ENST00000274289.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 57754916: mut_end: 57754916: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G92S: mutation info source: CCLE: ref_target(−10 + 10): TTTGCAAAGC C ACCCTGAAAG (SEQ ID NO: 729): mut_target(−10 + 10): TTTGCAAAGC T ACCCTGAAAG (SEQ ID NO: 730): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_ dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-228: [crRNA sequence]: crRNA sequence: TTTCCTTTCAGGGT A GCTTTGCAAAATG (SEQ ID NO: 733): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTCAGGGTAGCTTTGCAAAATG (SEQ ID NO: 734): [Target gene information]: Gene ID: 10769: Symbol: PLK2: Ensembl_ Transcript_ID: ENST00000274289.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 57754916: mut_end: 57754916: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G92S: mutation info source: CCLE: ref_target(−10 + 10): TTTGCAAAGC C ACCCTGAAAG (SEQ ID NO: 729): mut_target(−10 + 10): TTTGCAAAGC T ACCCTGAAAG (SEQ ID NO: 730): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-229: [crRNA sequence]: crRNA sequence: TTTCAGGGT A GCTTTGCAAAATGTTACG (SEQ ID NO: 735): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGGTAGCTTTGCAAAATGTTACG (SEQ ID NO: 736): [Target gene information]: Gene ID: 10769: Symbol: PLK2: Ensembl_ Transcript_ID: ENST00000274289.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 57754916: mut_end: 57754916: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G92S: mutation info source: CCLE: ref_target(−10 + 10): TTTGCAAAGC C ACCCTGAAAG (SEQ ID NO: 729): mut_target(−10 + 10): TTTGCAAAGC T ACCCTGAAAG (SEQ ID NO: 730): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 6: indel length: 0: CRISPR gRNA ID: GF-CCELg12-230: [crRNA sequence]: crRNA sequence: TTTGTTTCCTTTCAGGGT A GCTTTGCAA (SEQ ID NO: 737): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTCCTTTCAGGGTAGCTTTGCAA (SEQ ID NO: 738): [Target gene information]: Gene ID: 10769: Symbol: PLK2: Ensembl_ Transcript_ID: ENST00000274289.3: GRCh: 37: Chr: 5: [Target cancer mutation information]: mut_start: 57754916: mut_end: 57754916: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.G92S: mutation info source: CCLE: ref_target(−10 + 10): TTTGCAAAGC C ACCCTGAAAG (SEQ ID NO: 729): mut_target(−10 + 10): TTTGCAAAGC T ACCCTGAAAG (SEQ ID NO: 730): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-231: [crRNA sequence]: crRNA sequence: TTTGGCGAAGGCCA A GAGGCCGCCCACC (SEQ ID NO: 739): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCGAAGGCCAAGAGGCCGCCCACC (SEQ ID NO: 740): [Target gene information]: Gene ID: 5424: Symbol: POLD1: Ensembl_ Transcript_ID: ENST00000440232.2: GRCh: 37: CHr: 19: [Target cancer mutation information]: mut_start: 50919920: mut_end: 50919920: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.L1003L: mutation info source: CCLE: ref_target(−10 + 10): GGGCGGCCTC C TGGCCTTCGC (SEQ ID NO: 741): mut_target(−10 + 10): GGGCGGCCTC T TGGCCTTCGC (SEQ ID NO: 742): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-232: [crRNA sequence]: crRNA sequence: TT T CAACAGTTGGAGATTGACCATTATG (SEQ ID NO: 743): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AACAGTTGGAGATTGACCATTATG (SEQ ID NO: 744): [Target gene information]: Gene ID: 5424: Symbol: POLD1: Ensembl_ Transcript_ID: ENST00000440232.2: GRCh: 37: Chr: 19: [Target cancer mutation information]: mut_start: 50902713: mut_end: 50902713: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.F96F: mutation info source: CCLE: ref_target(−10 + 10): CCCTCATCTT C CAACAGTTGG (SEQ ID NO: 745): mut_target(−10 + 10): CCCTCATCTT T CAACAGTTGG (SEQ ID NO: 746): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-233: [crRNA sequence]: crRNA sequence: TTTCACTCAGGGATGAT GC GCCACTTCC (SEQ ID NO: 747): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACTCAGGGATGATGCGCCACTTCC (SEQ ID NO: 748): [Target gene information]: Gene ID: 5426: Symbol: POLE: Ensembl_ Transcript_ID: ENST00000320574.5: GRCh: 37: CHr: 12: [Target cancer mutation information]: mut_start: 133257857: mut_end: 133257857: mut_class: Frame_Shift_Del: mut_type: DEL: ref_seq: C: mut_seq: -: mut_aa: p.G24fs: mutation info source: CCLE: ref_target(−10 + 10): GGAAGTGGCG C CATCATCCCT (SEQ ID NO: 749): mut_target(−10 + 10): GGAAGTGGCG-CATCATCCCT (SEQ ID NO: 750): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 15: indel length: 1: CRISPR gRNA ID: GF-CCELg12-234: [crRNA sequence]: crRNA sequence: TTTGCCCGGGAGCACTTGTGGGG G TTCA (SEQ ID NO: 751): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCCGGGAGCACTTGTGGGGGTTCA (SEQ ID NO: 752): [Target gene information]: Gene ID: 8493: Symbol: PPM1D: Ensembl_ Transcript_ID: ENST00000305921.3: GRCh: 37: CHr: 17: [Target cancer mutation information]: mut_start: 58678141: mut_end: 58678141: mut_class: Silent: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.G122G: mutation info source: CCLE: ref_target(−10 + 10): ACTTGTGGGG T TTCATCAAGA (SEQ ID NO: 753): mut_target(−10 + 10): ACTTGTGGGG G TTCATCAAGA (SEQ ID NO: 754): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_ dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-235: [crRNA sequence]: crRNA sequence: TTTCCA T TGCAACTCCCGCCACGTGATT (SEQ ID NO: 755): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CATTGCAACTCCCGCCACGTGATT (SEQ ID NO: 756): [Target gene information]: Gene ID: 5071: Symbol: PARK2: Ensembl_ Transcript_ID: ENST00000366898.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 162206919: mut_end: 162206919: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.Q252H: mutation info source: CCLE: ref_target(−10 + 10): GGGAGTTGCA C TGGAAAACCA (SEQ ID NO: 757): mut_target(−10 + 10): GGGAGTTGCA A TGGAAAACCA (SEQ ID NO: 758): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-236: [crRNA sequence]: crRNA sequence: TTTTCCA T TGCAACTCCCGCCACGTGAT (SEQ ID NO: 759): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCATTGCAACTCCCGCCACGTGAT (SEQ ID NO: 760): [Target gene information]: Gene ID: 5071: Symbol: PARK2: Ensembl_ Transcript_ID: ENST00000366898.1: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 162206919: mut_end: 162206919: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.Q252H: mutation info source: CCLE: ref_target(−10 + 10): GGGAGTTGCA C TGGAAAACCA (SEQ ID NO: 757): mut_target(−10 + 10): GGGAGTTGCA A TGGAAAACCA (SEQ ID NO: 758): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-237: [crRNA sequence]: crRNA sequence: TTTG T TTGAATTTTTGTTGGGGGCTGTG (SEQ ID NO: 761): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTGAATTTTTGTTGGGGGCTGTG (SEQ ID NO: 762): [Target gene information]: Gene ID: 5727: Symbol: PTCH1: Ensembl_ Transcript_ID: ENST00000331920.6: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 98242676: mut_end: 98242676: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.T314N: mutation info source: CCLE: ref_target(−10 + 10): ACTCACTTTG G TTGAATTTTT (SEQ ID NO: 763): mut_target(−10 + 10): ACTCACTTTG T TTGAATTTTT (SEQ ID NO: 764): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-238: [crRNA sequence]: crRNA sequence:  T TTGAATTTTTGTTGGGGGCTGTGGCGG (SEQ ID NO: 765): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATTTTTGTTGGGGGCTGTGGCGG (SEQ ID NO: 766): [Target gene information]: Gene ID: 5727: Symbol: PTCH1: Ensembl_ Transcript_ID: ENST00000331920.6: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 98242676: mut_end: 98242676: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.T314N: mutation info source: CCLE: ref_target(−10 + 10): ACTCACTTTG G TTGAATTTTT (SEQ ID NO: 763): mut_target(−10 + 10): ACTCACTTTG T TTGAATTTTT (SEQ ID NO: 764): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-239: [crRNA sequence]: crRNA sequence: TTTG T TTGAATTTTTGTTGGGGGCTGTG (SEQ ID NO: 761): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTGAATTTTTGTTGGGGGCTGTG (SEQ ID NO: 762): [Target gene information]: Gene ID: 5727: Symbol: PTCH1: Ensembl_ Transcript_ID: ENST00000331920.6: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 98242676: mut_end: 98242676: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.T314N: mutation info source: CCLE: ref_target(−10 + 10): ACTCACTTTG G TTGAATTTTT (SEQ ID NO: 763): mut_target(−10 + 10): ACTCACTTTG T TTGAATTTTT (SEQ ID NO: 764): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-240: [crRNA sequence]: crRNA sequence:  T TTGAATTTTTGTTGGGGGCTGTGGCGG (SEQ ID NO: 765): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATTTTTGTTGGGGGCTGTGGCGG (SEQ ID NO: 766): [Target gene information]: Gene ID: 5727: Symbol: PTCH1: Ensembl_ Transcript_ID: ENST00000331920.6: GRCh: 37: CHr: 9: [Target cancer mutation information]: mut_start: 98242676: mut_end: 98242676: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.T314N: mutation info source: CCLE: ref_target(−10 + 10): ACTCACTTTG G TTGAATTTTT (SEQ ID NO: 763): mut_target(−10 + 10): ACTCACTTTG T TTGAATTTTT (SEQ ID NO: 764): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-241: [crRNA sequence]: crRNA sequence:  T TTTTTTGAATGTAACAACCCAGTTTAA (SEQ ID NO: 767): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTGAATGTAACAACCCAGTTTAA (SEQ ID NO: 768): [Target gene information]: Gene ID: 5727: Symbol: PTCH1: Ensembl_ Transcript_ID: ENST00000331920.6: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 98268792: mut_end: 98268793: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: T: mut_aa: p.N97fs: mutation info source: CCLE: ref_target(−10 + 10): ACTTGCCGCA-TTTTTTGAAT (SEQ ID NO: 769): mut_target(−10 + 10): ACTTGCCGCA T TTTTTTGAAT (SEQ ID NO: 770): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: −3: indel length: 1: CRISPR gRNA ID: GF-CCELg12-242: [crRNA sequence]: crRNA sequence: TTTCTATGGGGAAGTAAG A ACCAGAGAC (SEQ ID NO: 771): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TATGGGGAAGTAAGAACCAGAGAC (SEQ ID NO: 772): [Target gene information]: Gene ID: 5728: Symbol: PTEN: Ensembl_ Transcript_ID: ENST00000371953.3: GRCh: 37: Chr: 10: [Target cancer mutation information]: mut_start: 89692993: mut_end: 89692993: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.R159R: mutation info source: CCLE: ref_target(−10 + 10): GGGAAGTAAG G ACCAGAGACA (SEQ ID NO: 773): mut_target(−10 + 10): GGGAAGTAAG A ACCAGAGACA (SEQ ID NO: 774): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-243: [crRNA sequence]: crRNA sequence: TTTGTCTCTGGT T CTTACTTCCCCATAG (SEQ ID NO: 775): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTCTGGTTCTTACTTCCCCATAG (SEQ ID NO: 776): [Target gene information]: Gene ID: 5728: Symbol: PTEN: Ensembl_ Transcript_ID: ENST00000371953.3: GRCh: 37: Chr: 10: [Target cancer mutation information]: mut_start: 89692993: mut_end: 89692993: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.R159R: mutation info source: CCLE: ref_target(−10 + 10): GGGAAGTAAG G ACCAGAGACA (SEQ ID NO: 773): mut_target(−10 + 10): GGGAAGTAAG A ACCAGAGACA (SEQ ID NO: 774): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-244: [crRNA sequence]: crRNA sequence: TTTTTGTCTCTGGT T CTTACTTCCCCAT (SEQ ID NO: 777): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTCTCTGGTTCTTACTTCCCCAT (SEQ ID NO: 778): [Target gene information]: Gene ID: 5728: Symbol: PTEN: Ensembl_ Transcript_ID: ENST00000371953.3: GRCh: 37: Chr: 10: [Target cancer mutation information]: mut_start: 89692993: mut_end: 89692993: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.R159R: mutation info source: CCLE: ref_target(−10 + 10): GGGAAGTAAG G ACCAGAGACA (SEQ ID NO: 773): mut_target(−10 + 10): GGGAAGTAAG A ACCAGAGACA (SEQ ID NO: 774): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-245: [crRNA sequence]: crRNA sequence: TTTTGTCTCTGGT T CTTACTTCCCCATA (SEQ ID NO: 779): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTCTCTGGTTCTTACTTCCCCATA (SEQ ID NO: 780): [Target gene information]: Gene ID: 5728: Symbol: PTEN: Ensembl_ Transcript_ID: ENST00000371953.3: GRCh: 37: Chr: 10: [Target cancer mutation information]: mut_start: 89692993: mut_end: 89692993: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.R159R: mutation info source: CCLE: ref_target(−10 + 10): GGGAAGTAAG G ACCAGAGACA (SEQ ID NO: 773): mut_target(−10 + 10): GGGAAGTAAG A ACCAGAGACA (SEQ ID NO: 774): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-246: [crRNA sequence]: crRNA sequence: TTTCGTTGTCATGACAATCACTC A GGAG (SEQ ID NO: 781): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTGTCATGACAATCACTCAGGAG (SEQ ID NO: 782): [Target gene information]: Gene ID: 5781: Symbol: PTPN11: Ensembl_ Transcript_ID: ENST00000351677.2: GRCh: 37: Chr: 12: [Target cancer mutation information]: mut_start: 112915778: mut_end: 112915778: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R351*: mutation info source: CCLE: ref_target(−10 + 10): AGAAAACTCC C GAGTGATTGT (SEQ ID NO: 783): mut_target(−10 + 10): AGAAAACTCC T GAGTGATTGT (SEQ ID NO: 784): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-247: [crRNA sequence]: crRNA sequence: TTT A GGTAAGGCTTGGATGGGGGAAGAT (SEQ ID NO: 785): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGTAAGGCTTGGATGGGGGAAGAT (SEQ ID NO: 786): [Target gene information]: Gene ID: 5789: Symbol: PTPRD: Ensembl_ Transcript_ID: ENST00000381196.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 8518422: mut_end: 8518422: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.P323P: mutation info source: CCLE: ref_target(−10 + 10): CTGGAGGTTT G GGTAAGGCTT (SEQ ID NO: 787): mut_target(−10 + 10): CTGGAGGTTT A GGTAAGGCTT (SEQ ID NO: 788): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-248: [crRNA sequence]: crRNA sequence: TTTATCTTCCCCCATCCAAGCCTTACC T (SEQ ID NO: 789): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTCCCCCATCCAAGCCTTACCT (SEQ ID NO: 790): [Target gene information]: Gene ID: 5789: Symbol: PTPRD: Ensembl_ Transcript_ID: ENST00000381196.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 8518422: mut_end: 8518422: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.P323P: mutation info source: CCLE: ref_target(−10 + 10): CTGGAGGTTT G GGTAAGGCTT (SEQ ID NO: 787): mut_target(−10 + 10): CTGGAGGTTT A GGTAAGGCTT (SEQ ID NO: 788): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-249: [crRNA sequence]: crRNA sequence: TTTGCTGCTGGATC G TCTCACACACACT (SEQ ID NO: 791): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGCTGGATCGTCTCACACACACT (SEQ ID NO: 792): [Target gene information]: Gene ID: 11122: Symbol: PTPRT: Ensembl_ Transcript_ID: ENST00000373187.1: GRCh: 37: Chr: 20: [Target cancer mutation information]: mut_start: 40710604: mut_end: 40710604: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.M1397T: mutation info source: CCLE: ref_target(−10 + 10): CTGCTGGATC A TCTCACACAC (SEQ ID NO: 793): mut_target(−10 + 10): CTGCTGGATC G TCTCACACAC (SEQ ID NO: 794): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-250: [crRNA sequence] : crRNA sequence: TTTTGCTGCTGGATC G TCTCACACACAC (SEQ ID NO: 795): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCTGCTGGATCGTCTCACACACAC (SEQ ID NO: 796): [Target gene information]: Gene ID: 11122: Symbol: PTPRT: Ensembl_ Transcript_ID: ENST00000373187.1: GRCh: 37: CHr: 20: [Target cancer mutation information]: mut_start: 40710604: mut_end: 40710604: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.M1397T: mutation info source: CCLE: ref_target(−10 + 10): CTGCTGGATC A TCTCACACAC (SEQ ID NO: 793): mut_target(−10 + 10): CTGCTGGATC G TCTCACACAC (SEQ ID NO: 794): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-251: [crRNA sequence]: crRNA sequence: TT T AGGGTCTGTGGGGCACAAAGGTGAA (SEQ ID NO: 797): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGGTCTGTGGGGCACAAAGGTGAA (SEQ ID NO: 798): [Target gene information]: Gene ID: 11122: Symbol: PTPRT: Ensembl_ Transcript_ID: ENST00000373187.1: GRCh: 37: CHr: 20: [Target cancer mutation information]: mut_start: 40730932: mut_end: 40730932: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L1182L: mutation info source: CCLE: ref_target(−10 + 10): TCACAATGTT G AGGGTCTGTG (SEQ ID NO: 799): mut_target(−10 + 10): TCACAATGTT T AGGGTCTGTG (SEQ ID NO: 800): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-252: [crRNA sequence]: crRNA sequence: TTTGTGCCCCACAGACCCT A AACATTGT (SEQ ID NO: 801): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGCCCCACAGACCCTAAACATTGT (SEQ ID NO: 802): [Target gene information]: Gene ID: 11122: Symbol: PTPRT: Ensembl_ Transcript_ID: ENST00000373187.1: GRCh: 37: Chr: 20: [Target cancer mutation information]: mut_start: 40730932: mut_end: 40730932: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.L1182L: mutation info source: CCLE: ref_target(−10 + 10): TCACAATGTT G AGGGTCTGTG (SEQ ID NO: 799): mut_target(−10 + 10): TCACAATGTT T AGGGTCTGTG (SEQ ID NO: 800): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-253: [crRNA sequence]: crRNA sequence: TTTCATGACTTTG T TCAGCCACTGCCTG (SEQ ID NO: 803): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATGACTTTGTTCAGCCACTGCCTG (SEQ ID NO: 804): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117874101: mut_end: 117874101: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.D118V: mutation info source: CCLE: ref_target(−10 + 10): CAGTGGCTGA T CAAAGTCATG (SEQ ID NO: 805): mut_target(−10 + 10): CAGTGGCTGA A CAAAGTCATG (SEQ ID NO: 806): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-254: [crRNA sequence]: crRNA sequence: TTTG T TCAGCCACTGCCTGACTTAGAGT (SEQ ID NO: 807): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCAGCCACTGCCTGACTTAGAGT (SEQ ID NO: 808): [Target gene information]: Gene ID: 5885: Symbol: RAD21:Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: CHr: 8: [Target cancer mutation information]: mut_start: 117874101: mut_end: 117874101: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.D118V: mutation info source: CCLE: ref_target(−10 + 10): CAGTGGCTGA T CAAAGTCATG (SEQ ID NO: 805): mut_target(−10 + 10): CAGTGGCTGA A CAAAGTCATG (SEQ ID NO: 806): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-255: [crRNA sequence]: crRNA sequence: TTTACCTGAAGAATTTCATGACTTTG T T (SEQ ID NO: 809): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCTGAAGAATTTCATGACTTTGTT (SEQ ID NO: 810): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117874101: mut_end: 117874101: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.D118V: mutation info source: CCLE: ref_target(−10 + 10): CAGTGGCTGA T CAAAGTCATG (SEQ ID NO: 805): mut_target(−10 + 10): CAGTGGCTGA A CAAAGTCATG (SEQ ID NO: 806): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-256: [crRNA sequence]: crRNA sequence: TTTCTTTTTTTTTTTTTTAATAGCT T TT (SEQ ID NO: 811): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTTTTTTTTTAATAGCTTTT (SEQ ID NO: 812): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: CHr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-257: [crRNA sequence]: crRNA sequence: TTTAATAGCT T TTTACACGCTGTCTTAC (SEQ ID NO: 815): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATAGCTTTTTACACGCTGTCTTAC (SEQ ID NO: 816): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-258: [crRNA sequence]: crRNA sequence: TTTTTCTTTTTTTTTTTTTTAATAGCT T (SEQ ID NO: 817): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTTTTTTTTTTTTTAATAGCTT (SEQ ID NO: 818): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-259: [crRNA sequence]: crRNA sequence: TTTTCTTTTTTTTTTTTTTAATAGCT T T (SEQ ID NO: 819): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTTTTTTTTTTTTAATAGCTTT (SEQ ID NO: 820): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-260: [crRNA sequence]: crRNA sequence: TTTTTTTTTTTTTTAATAGCT T TTTACA (SEQ ID NO: 821): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTTTTTAATAGCTTTTTACA (SEQ ID NO: 822): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-261: [crRNA sequence]: crRNA sequence: TTTTTTTTTTTTTAATAGCT T TTTACAC (SEQ ID NO: 823): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTTTTAATAGCTTTTTACAC (SEQ ID NO: 824): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 17: indel length: 0: CRISPR gRNA ID: GF-CCELg12-262: [crRNA sequence]: crRNA sequence: TTTTTTTTTTTTAATAGCT T TTTACACG (SEQ ID NO: 825): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTTTAATAGCTTTTTACACG (SEQ ID NO: 826): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-263: [crRNA sequence]: crRNA sequence: TTTTTTTTTTTAATAGCT T TTTACACGC (SEQ ID NO: 827): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTTAATAGCTTTTTACACGC (SEQ ID NO: 828): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-264: [crRNA sequence]: crRNA sequence: TTTTTTTTTTAATAGCT T TTTACACGCT (SEQ ID NO: 829): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTTAATAGCTTTTTACACGCT (SEQ ID NO: 830): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-265: [crRNA sequence]: crRNA sequence: TTTTTTTTTAATAGCT T TTTACACGCTG (SEQ ID NO: 831): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTAATAGCTTTTTACACGCTG (SEQ ID NO: 832): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq:  A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-266: [crRNA sequence]: crRNA sequence: TTTTTTTTAATAGCT T TTTACACGCTGT (SEQ ID NO: 833): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTAATAGCTTTTTACACGCTGT (SEQ ID NO: 834): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-267: [crRNA sequence]: crRNA sequence: TTTTTTTAATAGCT T TTTACACGCTGTC (SEQ ID NO: 835): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTAATAGCTTTTTACACGCTGTC (SEQ ID NO: 836): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-268: [crRNA sequence]: crRNA sequence: TTTTTTAATAGCT T TTTACACGCTGTCT (SEQ ID NO: 837): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAATAGCTTTTTACACGCTGTCT (SEQ ID NO: 838): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-269: [crRNA sequence]: crRNA sequence: TTTTTAATAGCT T TTTACACGCTGTCTT (SEQ ID NO: 839): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TAATAGCTTTTTACACGCTGTCTT (SEQ ID NO: 840): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-270: [crRNA sequence]: crRNA sequence: TTTTAATAGCT T TTTACACGCTGTCTTA (SEQ ID NO: 841): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AATAGCTTTTTACACGCTGTCTTA (SEQ ID NO: 842): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-271: [crRNA sequence]: crRNA sequence: T T TTTACACGCTGTCTTACACCGCTTGT (SEQ ID NO: 843): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TACACGCTGTCTTACACCGCTTGT (SEQ ID NO: 844): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-272: [crRNA sequence]: crRNA sequence:  T TTTACACGCTGTCTTACACCGCTTGTA (SEQ ID NO: 845): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACACGCTGTCTTACACCGCTTGTA (SEQ ID NO: 846): [Target gene information]: Gene ID: 5885: Symbol: RAD21: Ensembl_ Transcript_ID: ENST00000297338.2: GRCh: 37: Chr: 8: [Target cancer mutation information]: mut_start: 117864945: mut_end: 117864945: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.L388L: mutation info source: CCLE: ref_target(−10 + 10): AGCGTGTAAA G AGCTATTAAA (SEQ ID NO: 813): mut_target(−10 + 10): AGCGTGTAAA A AGCTATTAAA (SEQ ID NO: 814): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: −3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-273: [crRNA sequence]: crRNA sequence: TTTCCAAGAACCAAG C TCTTCCGAGGGA (SEQ ID NO: 847): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAAGAACCAAGCTCTTCCGAGGGA (SEQ ID NO: 848): [Target gene information]: Gene ID: 5979: Symbol: RET: Ensembl_ Transcript_ID: ENST00000355710.3: GRCh: 37: CHr: 10: [Target cancer mutation information]: mut_start: 43612063: mut_end: 43612063: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.N7235: mutation info source: CCLE: ref_target(−10 + 10): CCTCGGAAGA A CTTGGTTCTT (SEQ ID NO: 849): mut_target(−10 + 10): CCTCGGAAGA G CTTGGTTCTT (SEQ ID NO: 850): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-274 [crRNA sequence]: crRNA sequence: TTTTTCCAAGAACCAAG C TCTTCCGAGG (SEQ ID NO: 851): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCCAAGAACCAAGCTCTTCCGAGG (SEQ ID NO: 852): [Target gene information]: Gene ID: 5979: Symbol: RET: Ensembl_ Transcript_ID: ENST00000355710.3: GRCh: 37: CHr: 10: [Target cancer mutation information]: mut_start: 43612063: mut_end: 43612063: mut class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.N7235: mutation info source: CCLE: ref_target(−10 + 10): CCTCGGAAGA A CTTGGTTCTT (SEQ ID NO: 849): mut_target(−10 + 10): CCTCGGAAGA G CTTGGTTCTT (SEQ ID NO: 850): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 14: indel length: 0: CRISPR gRNA ID: GF-CCELg12-275: [crRNA sequence]: crRNA sequence: TTTTCCAAGAACCAAG C TCTTCCGAGGG (SEQ ID NO: 853): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCAAGAACCAAGCTCTTCCGAGGG (SEQ ID NO: 854): [Target gene information]: Gene ID: 5979: Symbol: RET: Ensembl_ Transcript_ID: ENST00000355710.3: GRCh: 37: CHr: 10: [Target cancer mutation information]: mut_start: 43612063: mut_end: 43612063: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.N7235: mutation info source: CCLE: ref_target(−10 + 10): CCTCGGAAGA A CTTGGTTCTT (SEQ ID NO: 849): mut_target(−10 + 10): CCTCGGAAGA G CTTGGTTCTT (SEQ ID NO: 850): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 13: indel length: 0: CRISPR gRNA ID: GF-CCELg12-276: [crRNA sequence]: crRNA sequence: TTTGGTTCTTGTACACAAACT A CATCAG (SEQ ID NO: 855): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTCTTGTACACAAACTACATCAG (SEQ ID NO: 856): [Target gene information]: Gene ID: 54894: Symbol: RNF43: Ensembl_ Transcript_ID: ENST00000584437.1: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 56440698: mut_end: 56440698: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.E174*: mutation info source: CCLE: ref_target(−10 + 10): TACACAAACT C CATCAGCTTC (SEQ ID NO: 857): mut_target(−10 + 10): TACACAAACT A CATCAGCTTC (SEQ ID NO: 858): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-277: [crRNA sequence]: crRNA sequence: TTTTGGTTCTTGTACACAAACT A CATCA (SEQ ID NO: 859): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGTTCTTGTACACAAACTACATCA (SEQ ID NO: 860): [Target gene information]: Gene ID: 54894: Symbol: RNF43: Ensembl_ Transcript_ID: ENST00000584437.1: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 56440698: mut_end: 56440698: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.E174*: mutation info source: CCLE: ref_target(−10 + 10): TACACAAACT C CATCAGCTTC (SEQ ID NO: 857): mut_target(−10 + 10): TACACAAACT A CATCAGCTTC (SEQ ID NO: 858): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_dist: 19: indel length: 0: CRISPR gRNA ID: GF-CCELg12-278: [crRNA sequence]: crRNA sequence: TTTCTTCTTATTTTAGATAAA T CGTTTC (SEQ ID NO: 861): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCTTATTTTAGATAAATCGTTTC (SEQ ID NO: 862): [Target gene information]: Gene ID: 6098: Symbol: ROS1: Ensembl_ Transcript_ID: ENST00000368508.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 117665420: mut_end: 117665420: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A14435: mutation info source: CCLE: ref_target(−10 + 10): GACAGAAACG C TTTATCTAAA (SEQ ID NO: 863): mut_target(−10 + 10): GACAGAAACG A TTTATCTAAA (SEQ ID NO: 864): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 18: indel length: 0: CRISPR gRNA ID: GF-CCELg12-279: [crRNA sequence]: crRNA sequence: TTTGGTTTCTTCTTATTTTAGATAAA T C (SEQ ID NO: 865): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTTCTTCTTATTTTAGATAAATC (SEQ ID NO: 866): [Target gene information]: Gene ID: 6098: Symbol: ROS1: Ensembl_ Transcript_ID: ENST00000368508.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 117665420: mut_end: 117665420: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A14435: mutation info source: CCLE: ref_target(−10 + 10): GACAGAAACG C TTTATCTAAA (SEQ ID NO: 863): mut_target(−10 + 10): GACAGAAACG A TTTATCTAAA (SEQ ID NO: 864): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 23: indel length: 0: CRISPR gRNA ID: GF-CCELg12-280: [crRNA sequence]: crRNA sequence: TTTAGATAAA T CGTTTCTGTCTCTAGCT (SEQ ID NO: 867): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GATAAATCGTTTCTGTCTCTAGCT (SEQ ID NO: 868): [Target gene information]: Gene ID: 6098: Symbol: ROS1: Ensembl_ Transcript_ID: ENST00000368508.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 117665420: mut_end: 117665420: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A14435: mutation info source: CCLE: ref_target(−10 + 10): GACAGAAACG C TTTATCTAAA (SEQ ID NO: 863): mut_target(−10 + 10): GACAGAAACG A TTTATCTAAA (SEQ ID NO: 864): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-281: [crRNA sequence]: crRNA sequence: TTTTGGTTTCTTCTTATTTTAGATAAA T (SEQ ID NO: 869): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGTTTCTTCTTATTTTAGATAAAT (SEQ ID NO: 870): [Target gene information]: Gene ID: 6098: Symbol: ROS1: Ensembl_ Transcript_ID: ENST00000368508.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 117665420: mut_end: 117665420: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A14435: mutation info source: CCLE: ref_target(−10 + 10): GACAGAAACG C TTTATCTAAA (SEQ ID NO: 863): mut_target(−10 + 10): GACAGAAACG A TTTATCTAAA (SEQ ID NO: 864): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_ dist: 24: indel length: 0: CRISPR gRNA ID: GF-CCELg12-282: [crRNA sequence]: crRNA sequence: TTTTAGATAAA T CGTTTCTGTCTCTAGC (SEQ ID NO: 871): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGATAAATCGTTTCTGTCTCTAGC (SEQ ID NO: 872): [Target gene information]: Gene ID: 6098: Symbol: ROS1: Ensembl_ Transcript_ID: ENST00000368508.3: GRCh: 37: Chr: 6: [Target cancer mutation information]: mut_start: 117665420: mut_end: 117665420: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.A14435: mutation info source: CCLE: ref_target(−10 + 10): GACAGAAACG C TTTATCTAAA (SEQ ID NO: 863): mut_target(−10 + 10): GACAGAAACG A TTTATCTAAA (SEQ ID NO: 864): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-283: [crRNA sequence]: crRNA sequence: TTTCTTGTTGGTATTTTT G TTGGTAACA (SEQ ID NO: 873): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGTTGGTATTTTTGTTGGTAACA (SEQ ID NO: 874): [Target gene information]: Gene ID: 23429: Symbol: RYBP: Ensembl_ Transcript_ID: ENST00000477973.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 72428425: mut_end: 72428425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E193Q: mutation info source: CCLE: ref_target(−10 + 10): TGGTATTTTT C TTGGTAACAC (SEQ ID NO: 875): mut_target(−10 + 10): TGGTATTTTT G TTGGTAACAC (SEQ ID NO: 876): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 15: indel length: 0: CRISPR gRNA ID: GF-CCELg12-284: [crRNA sequence]: crRNA sequence: TTT G TTGGTAACACTAGGACTAATTTCC (SEQ ID NO: 877): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGGTAACACTAGGACTAATTTCC (SEQ ID NO: 878): [Target gene information]: Gene ID: 23429: Symbol: RYBP: Ensembl_ Transcript_ID: ENST00000477973.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 72428425: mut_end: 72428425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E193Q: mutation info source: CCLE: ref_target(−10 + 10): TGGTATTTTT C TTGGTAACAC (SEQ ID NO: 875): mut_target(−10 + 10): TGGTATTTTT G TTGGTAACAC (SEQ ID NO: 876): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 0: indel length: 0: CRISPR gRNA ID: GF-CCELg12-285: [crRNA sequence]: crRNA sequence: TTTTCTTGTTGGTATTTTT G TTGGTAAC (SEQ ID NO: 879): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGTTGGTATTTTTGTTGGTAAC (SEQ ID NO: 880): [Target gene information]: Gene ID: 23429: Symbol: RYBP: Ensembl_ Transcript_ID: ENST00000477973.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 72428425: mut_end: 72428425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E193Q: mutation info source: CCLE: ref_target(−10 + 10): TGGTATTTTT C TTGGTAACAC (SEQ ID NO: 875): mut_target(−10 + 10): TGGTATTTTT G TTGGTAACAC (SEQ ID NO: 876): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 16: indel length: 0: CRISPR gRNA ID: GF-CCELg12-286: [crRNA sequence]: crRNA sequence: TTTTT G TTGGTAACACTAGGACTAATTT (SEQ ID NO: 881): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGTTGGTAACACTAGGACTAATTT (SEQ ID NO: 882): [Target gene information]: Gene ID: 23429: Symbol: RYBP: Ensembl_ Transcript_ID: ENST00000477973.2: GRCh: 37: Chr: 3: [Target cancer mutation information]: mut_start: 72428425: mut_end: 72428425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E193Q: mutation info source: CCLE: ref_target(−10 + 10): TGGTATTTTT C TTGGTAACAC (SEQ ID NO: 875): mut_target(−10 + 10): TGGTATTTTT G TTGGTAACAC (SEQ ID NO: 876): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_dist: 2: indel length: 0: CRISPR gRNA ID: GF-CCELg12-287: [crRNA sequence]: crRNA sequence: TTTT G TTGGTAACACTAGGACTAATTTC (SEQ ID NO: 883): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GTTGGTAACACTAGGACTAATTTC (SEQ ID NO: 884): [Target gene information]: Gene ID: 23429: Symbol: RYBP: Ensembl_ Transcript_ID: ENST00000477973.2: GRCh: 37: CHr: 3: [Target cancer mutation information]: mut_start: 72428425: mut_end: 72428425: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: G: mut_aa: p.E193Q: mutation info source: CCLE: ref_target(−10 + 10): TGGTATTTTT C TTGGTAACAC (SEQ ID NO: 875): mut_target(−10 + 10): TGGTATTTTT G TTGGTAACAC (SEQ ID NO: 876): [Model Cell line information]: cell: NCIH1563: cancer_type: LUNG: PAM_ dist: 1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-288: [crRNA sequence]: crRNA sequence: TTTCTT T ATGGGGCTTTCAGGCAGACAT (SEQ ID NO: 885): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTATGGGGCTTTCAGGCAGACAT (SEQ ID NO: 886): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 3: indel length: 0: CRISPR gRNA ID: GF-CCELg12-289: [crRNA sequence]: crRNA sequence: TTTATTTTTCTT T ATGGGGCTTTCAGGC (SEQ ID NO: 889): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTTTTCTTTATGGGGCTTTCAGGC (SEQ ID NO: 890): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-290: [crRNA sequence]: crRNA sequence: TT T ATGGGGCTTTCAGGCAGACATCAGG (SEQ ID NO: 891): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGGGGCTTTCAGGCAGACATCAGG (SEQ ID NO: 892): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-291: [crRNA sequence]: crRNA sequence: TTTTATTTTTCTT T ATGGGGCTTTCAGG (SEQ ID NO: 893): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ATTTTTCTTTATGGGGCTTTCAGG (SEQ ID NO: 894): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 10: indel length: 0: CRISPR gRNA ID: GF-CCELg12-292: [crRNA sequence]: crRNA sequence: TTTTTCTT T ATGGGGCTTTCAGGCAGAC (SEQ ID NO: 895): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTTATGGGGCTTTCAGGCAGAC (SEQ ID NO: 896): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-293: [crRNA sequence]: crRNA sequence: TTTTCTT T ATGGGGCTTTCAGGCAGACA (SEQ ID NO: 897): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTTATGGGGCTTTCAGGCAGACA (SEQ ID NO: 898): [Target gene information]: Gene ID: 4068: Symbol: SH2D1A: Ensembl_ Transcript_ID: ENST00000371139.4: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123505240: mut_end: 123505240: mut_class: Silent: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.*129*: mutation info source: CCLE: ref_target(−10 + 10): AAAGCCCCAT G AAGAAAAATA (SEQ ID NO: 887): mut_target(−10 + 10): AAAGCCCCAT A AAGAAAAATA (SEQ ID NO: 888): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-294: [crRNA sequence]: crRNA sequence: TTTGGAG A GGCTGGTGTCCCTGTACAAC (SEQ ID NO: 899): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGAGGCTGGTGTCCCTGTACAAC (SEQ ID NO: 900): [Target gene information]: Gene ID: 6597: Symbol: SMARCA4: Ensembl_ Transcript_ID: ENST00000429416.3: GRCh: 37: Chr: 19: [Target cancer mutation information]: mut_start: 11123640: mut_end: 11123640: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: A: mut_aa: p.W764R: mutation info source: CCLE: ref_target(−10 + 10): AGGTTTGGAG T GGCTGGTGTC (SEQ ID NO: 901): mut_target(−10 + 10): AGGTTTGGAG A GGCTGGTGTC (SEQ ID NO: 902): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 4: indel length: 0: CRISPR gRNA ID: GF-CCELg12-295: [crRNA sequence]: crRNA sequence: TTTCTGCCCAGTGCACCGGCCCC A GTGG (SEQ ID NO: 903): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGCCCAGTGCACCGGCCCCAGTGG (SEQ ID NO: 904): [Target gene information]: Gene ID: 6608: Symbol: SMO: Ensembl_ Transcript_ID: ENST00000249373.3: GRCh: 37: CHr: 7: [Target cancer mutation information]: mut_start: 128852193: mut_end: 128852193: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.P755P: mutation info source: CCLE: ref_target(−10 + 10): CACCGGCCCC C GTGGCATGGG (SEQ ID NO: 905): mut_target(−10 + 10): CACCGGCCCC A GTGGCATGGG (SEQ ID NO: 906): [Model Cell line information]: cell: NCIH2126: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-296: [crRNA sequence]: crRNA sequence: TTTGTCTCCA T GGGAGTGGAAGGCGCCT (SEQ ID NO: 907): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTCCATGGGAGTGGAAGGCGCCT (SEQ ID NO: 908): [Target gene information]: Gene ID: 51684: Symbol: SUFU: Ensembl_ Transcript_ID: ENST00000369902.3: GRCh: 37: CHr: 10: [Target cancer mutation information]: mut_start: 104377121: mut_end: 104377121: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.T411M: mutation info source: CCLE: ref_target(−10 + 10): TTTGTCTCCA C GGGAGTGGAA (SEQ ID NO: 909): mut_target(−10 + 10): TTTGTCTCCA T GGGAGTGGAA (SEQ ID NO: 910): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_ dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-297: [crRNA sequence]: crRNA sequence: TTTCTTGGCTTACCCCGAAGTTAC A TCT (SEQ ID NO: 911): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGGCTTACCCCGAAGTTACATCT (SEQ ID NO: 912): [Target gene information]: Gene ID: 54790: Symbol: TET2: Ensembl_ Transcript_ID: ENST00000540549.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 106155466: mut_end: 106155466: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R123C: mutation info source: CCLE: ref_target(−10 + 10): TGGAGAAAGA C GTAACTTCGG (SEQ ID NO: 913): mut_target(−10 + 10): TGGAGAAAGA T GTAACTTCGG (SEQ ID NO: 914): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-298: [crRNA sequence]: crRNA sequence: TTTAGCATTGCAGCT C GTTTACTGGCAT (SEQ ID NO: 915): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GCATTGCAGCTCGTTTACTGGCAT (SEQ ID NO: 916): [Target gene information]: Gene ID: 54790: Symbol: TET2: Ensembl_ Transcript_ID: ENST00000540549.1: GRCh: 37: Chr: 4: [Target cancer mutation information]: mut_start: 106156019: mut_end: 106156019: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: T: mut_seq: G: mut_aa: p.L307R: mutation info source: CCLE: ref_target(−10 + 10): GCCAGTAAAC T AGCTGCAATG (SEQ ID NO: 917): mut_target(−10 + 10): GCCAGTAAAC G AGCTGCAATG (SEQ ID NO: 918): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 12: indel length: 0: CRISPR gRNA ID: GF-CCELg12-299: [crRNA sequence]: crRNA sequence: TTTCTTAATCC A CAATGCTGTAAGCCTA (SEQ ID NO: 919): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTAATCCACAATGCTGTAAGCCTA (SEQ ID NO: 920): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 101911534: mut_end: 101911534: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R487W: mutation info source: CCLE: ref_target(−10 + 10): TACAGCATTG C GGATTAAGAA (SEQ ID NO: 921): mut_target(−10 + 10): TACAGCATTG T GGATTAAGAA (SEQ ID NO: 922): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-300: [crRNA sequence]: crRNA sequence: TTTTCTTAATCC A CAATGCTGTAAGCCT (SEQ ID NO: 923): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTAATCCACAATGCTGTAAGCCT (SEQ ID NO: 924): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 101911534: mut_end: 101911534: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R487W: mutation info source: CCLE: ref_target(−10 + 10): TACAGCATTG C GGATTAAGAA (SEQ ID NO: 921): mut_target(−10 + 10): TACAGCATTG T GGATTAAGAA (SEQ ID NO: 922): [Model Cell line information]: cell: NCIH1437: cancer_type: LUNG: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-301: [crRNA sequence]: crRNA sequence: TTTGAAATCAAAGA G TATCTTGGTAAAG (SEQ ID NO: 925): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAATCAAAGAGTATCTTGGTAAAG (SEQ ID NO: 926): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 101907053: mut_end: 101907053: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.N3385: mutation info source: CCLE: ref_target(−10 + 10): AAATCAAAGA A TATCTTGGTA (SEQ ID NO: 927): mut_target(−10 + 10): AAATCAAAGA G TATCTTGGTA (SEQ ID NO: 928): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_ dist: 11: indel length: 0: CRISPR gRNA ID: GF-CCELg12-302: [crRNA sequence]: crRNA sequence: TTTACCAAGATA C TCTTTGATTTCAAAT (SEQ ID NO: 929): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CCAAGATACTCTTTGATTTCAAAT (SEQ ID NO: 930): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 101907053: mut_end: 101907053: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: G: mut_aa: p.N3385: mutation info source: CCLE: ref_target(−10 + 10): AAATCAAAGA A TATCTTGGTA (SEQ ID NO: 927): mut_target(−10 + 10): AAATCAAAGA G TATCTTGGTA (SEQ ID NO: 928): [Model Cell line information]: cell: HPAFII: cancer_type: PANCREAS: PAM_dist: 9: indel length: 0: CRISPR gRNA ID: GF-CCELg12-303: [crRNA sequence]: crRNA sequence: TTTACAGACAATGGTACTTGGACT T AGC (SEQ ID NO: 931): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CAGACAATGGTACTTGGACTTAGC (SEQ ID NO: 932): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 101904835: mut_end: 101904835: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q275*: mutation info source: CCLE: ref_target(−10 + 10): TACTTGGACT C AGCTCTGGTT (SEQ ID NO: 933): mut_target(−10 + 10): TACTTGGACT T AGCTCTGGTT (SEQ ID NO: 934): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 21: indel length: 0: CRISPR gRNA ID: GF-CCELg12-304 [crRNA sequence]: crRNA sequence: TTTTACAGACAATGGTACTTGGACT T AG (SEQ ID NO: 935): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat ACAGACAATGGTACTTGGACTTAG (SEQ ID NO: 936): [Target gene information]: Gene ID: 7046: Symbol: TGFBR1: Ensembl_ Transcript_ID: ENST00000374994.4: GRCh: 37: CHr: 9: [Target cancer mutation information]: mut_start: 101904835: mut_end: 101904835: mut_class: Nonsense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.Q275*: mutation info source: CCLE: ref_target(−10 + 10): TACTTGGACT C AGCTCTGGTT (SEQ ID NO: 933): mut_target(−10 + 10): TACTTGGACT T AGCTCTGGTT (SEQ ID NO: 934): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-305: [crRNA sequence]: crRNA sequence: TTTCGACATA T TGTGGTGGTGCCCTATG (SEQ ID NO: 90): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GACATATTGTGGTGGTGCCCTATG (SEQ ID NO: 99): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7578205: mut_end: 7578205: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.S215I: mutation info source: CCLE: ref_target(−10 + 10): CACCACCACA C TATGTCGAAA (SEQ ID NO: 93): mut_target(−10 + 10): CACCACCACA A TATGTCGAAA (SEQ ID NO: 96): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-306: [crRNA sequence]: crRNA sequence: TTTTCGACATA T TGTGGTGGTGCCCTAT (SEQ ID NO: 937): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGACATATTGTGGTGGTGCCCTAT (SEQ ID NO: 938): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7578205: mut_end: 7578205: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: A: mut_aa: p.S215I: mutation info source: CCLE: ref_target(−10 + 10): CACCACCACA C TATGTCGAAA (SEQ ID NO: 93): mut_target(−10 + 10): CACCACCACA A TATGTCGAAA (SEQ ID NO: 96): [Model Cell line information]: cell: NCIH661: cancer_type: LUNG: PAM_dist: 8: indel length: 0: CRISPR gRNA ID: GF-CCELg12-307: [crRNA sequence]: crRNA sequence: TTTGAGGTGC A TGTTTGTGCCTGTCCTG (SEQ ID NO: 939): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AGGTGCATGTTTGTGCCTGTCCTG (SEQ ID NO: 940): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7577120: mut_end: 7577120: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.R273H: mutation info source: CCLE: ref_target(−10 + 10): GGCACAAACA C GCACCTCAAA (SEQ ID NO: 941): mut_target(−10 + 10): GGCACAAACA T GCACCTCAAA (SEQ ID NO: 942): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_ dist: 7: indel length: 0: CRISPR gRNA ID: GF-CCELg12-308: [crRNA sequence]: crRNA sequence: TTTCGACATAGTGTGGT GC CCTATGAGC (SEQ ID NO: 943): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GACATAGTGTGGTGCCCTATGAGC (SEQ ID NO: 944): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: CHr: 17: [Target cancer mutation information]: mut_start: 7578195: mut_end: 7578197: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: CAC: mut_seq: -: mut_aa: p.V218del: mutation info source: CCLE: ref_target(−10 + 10): GCTCATAGGG CAC CACCACACTA (SEQ ID NO: 945): mut_target(−10 + 10): GCTCATAGGG---CACCACACTA (SEQ ID NO: 946): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_dist: 15: indel length: 3: CRISPR gRNA ID: GF-CCELg12-309: [crRNA sequence]: crRNA sequence: TTTTCGACATAGTGTGGT GC CCTATGAG (SEQ ID NO: 947): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGACATAGTGTGGTGCCCTATGAG (SEQ ID NO: 948): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: CHr: 17: [Target cancer mutation information]: mut_start: 7578195: mut_end: 7578197: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: CAC: mut_seq: -: mut_aa: p.V218del: mutation info source: CCLE: ref_target(−10 + 10): GCTCATAGGG CAC CACCACACTA (SEQ ID NO: 945): mut_target(−10 + 10): GCTCATAGGG---CACCACACTA (SEQ ID NO: 946): [Model Cell line information]: cell: HCC827GR5: cancer_type: LUNG: PAM_ dist: 16: indel length: 3: CRISPR gRNA ID: GF-CCELg12-310: [crRNA sequence]: crRNA sequence: TTTCGACATAGTGTGGT GC CCTATGAGC (SEQ ID NO: 943): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GACATAGTGTGGTGCCCTATGAGC (SEQ ID NO: 944): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7578195: mut_end: 7578197: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: CAC: mut_seq: -: mut_aa: p.V218del: mutation info source: CCLE: ref_target(−10 + 10): GCTCATAGGG CAC CACCACACTA (SEQ ID NO: 945): mut_target(−10 + 10): GCTCATAGGG---CACCACACTA (SEQ ID NO: 946): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: 15: indel length: 3: CRISPR gRNA ID: GF-CCELg12-311: [crRNA sequence]: crRNA sequence: TTTTCGACATAGTGTGGT GC CCTATGAG (SEQ ID NO: 947): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CGACATAGTGTGGTGCCCTATGAG (SEQ ID NO: 948): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7578195: mut_end: 7578197: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: CAC: mut_seq: -: mut_aa: p.V218del: mutation info source: CCLE: ref_target(−10 + 10): GCTCATAGGG CAC CACCACACTA (SEQ ID NO: 945): mut_target(−10 + 10): GCTCATAGGG---CACCACACTA (SEQ ID NO: 946): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: 16: indel length: 3: CRISPR gRNA ID: GF-CCELg12-312: [crRNA sequence]: crRNA sequence: TTTCCTTCCACTCGGATAAGATGC TG AG (SEQ ID NO: 949): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTCCACTCGGATAAGATGCTGAG (SEQ ID NO: 950): [Target gene information]: Gene ID: 7157: Symbol: TP53: Ensembl_ Transcript_ID: ENST00000269305.4: GRCh: 37: Chr: 17: [Target cancer mutation information]: mut_start: 7578275: mut_end: 7578277: mut_class: In_Frame_Del: mut_type: DEL: ref_seq: GAG: mut_seq: -: mut_aa: p.P191del: mutation info source: CCLE: ref_target(−10 + 10): ATAAGATGCT GAG GAGGGGCCAG (SEQ ID NO: 951): mut_target(−10 + 10): ATAAGATGCT---GAGGGGCCAG (SEQ ID NO: 952): [Model Cell line information]: cell: SW1990: cancer_type: PANCREAS: PAM_dist: 22: indel length: 3: CRISPR gRNA ID: GF-CCELg12-313: [crRNA sequence]: crRNA sequence: TTTGCTGGCTGGAAAGGGGTGACTC G CT (SEQ ID NO: 953): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTGGCTGGAAAGGGGTGACTCGCT (SEQ ID NO: 954): [Target gene information]: Gene ID: 7186: Symbol: TRAF2: Ensembl_ Transcript_ID: ENST00000536468.1: GRCh: 37: Chr: 9: [Target cancer mutation information]: mut_start: 139777164: mut_end: 139777164: mut_class: Splice_Site: mut_type: SNP: ref_seq: T: mut_seq: C: mut_aa: FALSE: mutation info source: CCLE: ref_target(−10 + 10): ATGGCCAGAG T GAGTCACCCC (SEQ ID NO: 955): mut_target(−10 + 10): ATGGCCAGAG C GAGTCACCCC (SEQ ID NO: 956): [Model Cell line information]: cell: NCIH1975: cancer_type: LUNG: PAM_dist: 22: indel length: 0: CRISPR gRNA ID: GF-CCELg12-314 [crRNA sequence]: crRNA sequence: TT T ATGGTTTCGGAGGCCCGACCGGGGC (SEQ ID NO: 957): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TGGTTTCGGAGGCCCGACCGGGGC (SEQ ID NO: 958): [Target gene information]: Gene ID: 7422: Symbol: VEGFA: Ensembl_ Transcript_ID: ENST00000523873.1: GRCh: 37: CHr: 6: [Target cancer mutation information]: mut_start: 43738986: mut_end: 43738986: mut_class: Start_Codon_SNP: mut_type: SNP: ref_seq: G: mut_seq: A: mut_aa: p.M1I: mutation info source: CCLE: ref_target(−10 + 10): CCGAAACCAT G AACTTTCTGC (SEQ ID NO: 959): mut_target(−10 + 10): CCGAAACCAT A AACTTTCTGC (SEQ ID NO: 960): [Model Cell line information]: cell: HCC827: cancer_type: LUNG: PAM_dist: −1: indel length: 0: CRISPR gRNA ID: GF-CCELg12-315: [crRNA sequence]: crRNA sequence: TTTCAAG A AAAAATTTTTATGTCTTAAT (SEQ ID NO: 961): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AAGAAAAAATTTTTATGTCTTAAT (SEQ ID NO: 962): [Target gene information]: Gene ID: 331: Symbol: XIAP: Ensembl_ Transcript_ID: ENST00000371199.3: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123041011: mut_end: 123041012: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q492fs: mutation info source: CCLE: ref_target(−10 + 10): TACTTTCAAG-AAAAATTTTT (SEQ ID NO: 963): mut_target(−10 + 10): TACTTTCAAG A AAAAATTTTT (SEQ ID NO: 964): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 4: indel length: 1: CRISPR gRNA ID: GF-CCELg12-316: [crRNA sequence]: crRNA sequence: TT T CTTGAAAGTAATGACTGTGTAGCAC (SEQ ID NO: 965): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTGAAAGTAATGACTGTGTAGCAC (SEQ ID NO: 966): [Target gene information]: Gene ID: 331: Symbol: XIAP: Ensembl_ Transcript_ID: ENST00000371199.3: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123041011: mut_end: 123041012: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q492fs: mutation info source: CCLE: ref_target(−10 + 10): TACTTTCAAG-AAAAATTTTT (SEQ ID NO: 963): mut_target(−10 + 10): TACTTTCAAG A AAAAATTTTT (SEQ ID NO: 964): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: −1: indel length: 1: CRISPR gRNA ID: GF-CCELg12-317: [crRNA sequence]: crRNA sequence: TTTTT T CTTGAAAGTAATGACTGTGTAG (SEQ ID NO: 967): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TTCTTGAAAGTAATGACTGTGTAG (SEQ ID NO: 968): [Target gene information]: Gene ID: 331: Symbol: XIAP: Ensembl_ Transcript_ID: ENST00000371199.3: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123041011: mut_end: 123041012: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q492fs: mutation info source: CCLE: ref_target(−10 + 10): TACTTTCAAG-AAAAATTTTT (SEQ ID NO: 963): mut_target(−10 + 10): TACTTTCAAG A AAAAATTTTT (SEQ ID NO: 964): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 2: indel length: 1: CRISPR gRNA ID: GF-CCELg12-318: [crRNA sequence]: crRNA sequence: TTTT T CTTGAAAGTAATGACTGTGTAGC (SEQ ID NO: 969): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat TCTTGAAAGTAATGACTGTGTAGC (SEQ ID NO: 970): [Target gene information]: Gene ID: 331: Symbol: XIAP: Ensembl_ Transcript_ID: ENST00000371199.3: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123041011: mut_end: 123041012: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q492fs: mutation info source: CCLE: ref_target(−10 + 10): TACTTTCAAG-AAAAATTTTT (SEQ ID NO: 963): mut_target(−10 + 10): TACTTTCAAG A AAAAATTTTT (SEQ ID NO: 964): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 1: indel length: 1: CRISPR gRNA ID: GF-CCELg12-319: [crRNA sequence]: crRNA sequence: TTT T CTTGAAAGTAATGACTGTGTAGCA (SEQ ID NO: 971): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat CTTGAAAGTAATGACTGTGTAGCA (SEQ ID NO: 972): [Target gene information]: Gene ID: 331: Symbol: XIAP: Ensembl_ Transcript_ID: ENST00000371199.3: GRCh: 37: Chr: X: [Target cancer mutation information]: mut_start: 123041011: mut_end: 123041012: mut_class: Frame_Shift_Ins: mut_type: INS: ref_seq: -: mut_seq: A: mut_aa: p.Q492fs: mutation info source: CCLE: ref_target(−10 + 10): TACTTTCAAG-AAAAATTTTT (SEQ ID NO: 963): mut_target(−10 + 10): TACTTTCAAG A AAAAATTTTT (SEQ ID NO: 964): [Model Cell line information]: cell: A549: cancer_type: LUNG: PAM_dist: 0: indel length: 1:: CRISPR gRNA ID: GF-CCELg12-320: [crRNA sequence]: crRNA sequence: TTTCAACT A TGTCAGTTTGTAATGGTAA (SEQ ID NO: 973): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat AACTATGTCAGTTTGTAATGGTAA (SEQ ID NO: 974): [Target gene information]: Gene ID: 7514: Symbol: XPO1: Ensembl_ Transcript_ID: ENST00000401558.2: GRCh: 37: CHr: 2: [Target cancer mutation information]: mut_start: 61726015: mut_end: 61726015: mut_class: Silent: mut_type: SNP: ref_seq: C: mut_seq: T: mut_aa: p.L208L: mutation info source: CCLE: ref_target(−10 + 10): CAAACTGACA C AGTTGAAATA (SEQ ID NO: 975): mut_target(−10 + 10): CAAACTGACA T AGTTGAAATA (SEQ ID NO: 976): [Model Cell line information]: cell: NCIH1573: cancer_type: LUNG: PAM_dist: 5: indel length: 0: CRISPR gRNA ID: GF-CCELg12-321: [crRNA sequence]: crRNA sequence: TTTGGAGATATGCTGTTGGGAAA C GATA (SEQ ID NO: 977): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GAGATATGCTGTTGGGAAACGATA (SEQ ID NO: 978): [Target gene information]: Gene ID: 463: Symbol: ZFHX3: Ensembl_ Transcript_ID: ENST00000268489.5: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 72827451: mut_end: 72827451: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: A: mut_seq: C: mut_aa: p.F3044V: mutation info source: CCLE: ref_target(−10 + 10): TGTTGGGAAA A GATATGGTCA (SEQ ID NO: 979): mut_target(−10 + 10): TGTTGGGAAA C GATATGGTCA (SEQ ID NO: 980): [Model Cell line information]: cell: NCIH1299: cancer_type: LUNG: PAM_dist: 20: indel length: 0: CRISPR gRNA ID: GF-CCELg12-322: [crRNA sequence]: crRNA sequence: TTTGGGACCCTCCACCGGGCTCGCC T GT (SEQ ID NO: 981): LbgRNA: AATTCTAATACGACTCACTATAGgtaatttctactaagtgtagat GGACCCTCCACCGGGCTCGCCTGT (SEQ ID NO: 982): [Target gene information]: Gene ID: 463: Symbol: ZFHX3: Ensembl_ Transcript_ID: ENST00000268489.5: GRCh: 37: Chr: 16: [Target cancer mutation information]: mut_start: 72821151: mut_end: 72821151: mut_class: Missense_Mutation: mut_type: SNP: ref_seq: G: mut_seq: T: mut_aa: p.P3675Q: mutation info source: CCLE: ref_target(−10 + 10): CGGGCTCGCC G GTCCGTCGGA (SEQ ID NO: 983): mut_target(−10 + 10): CGGGCTCGCC T GTCCGTCGGA (SEQ ID NO: 984): [Model Cell line information]: cell: NCIH460: cancer_type: LUNG: PAM_dist: 22: indel length: 0::

Table 7 and paragraph [00305] lists illustrative guide RNA sequences, determined using the methods described in Example 10, which can be used to guide a polypeptide comprising a Cas9 nuclease domain (e.g., a chimeric polypeptide comprising a Cas9 nuclease) to a target nucleic acid associated with a cancer, for example, lung cancer or pancreatic cancer. Variations from a reference sequence (e.g., wild type, healthy, or non-cancerous) are shown in underlined text.

Lengthy table referenced here US20220313796A1-20221006-T00001 Please refer to the end of the specification for access instructions.

Example 12. In Vivo Cell Targeting for Lung Cancer

An individual is identified as suffering from lung cancer. Sequence information associated with the individual's lung cancer cells is determined and mutations corresponding with the lung cancer are identified.

A guide RNA comprising a sequence from Table 6 and paragraph [00303] is then synthesized and is assembled into a ribonucleoprotein complex with a polypeptide comprising Cas12a nuclease domain. Alternatively, or in combination, a guide RNA comprising a sequence from Table 7 and paragraph [00305] is synthesized and is assembled into a ribonucleoprotein complex with a polypeptide comprising a Cas9 nuclease domain. The complex, comprising Cas12a, Cas9, or both, are then administered to the individual and symptom amelioration and tumor size reduction is observed.

Example 12. In Vivo Cell Targeting for Pancreatic Cancer

An individual is identified as suffering from pancreatic cancer. Sequence information associated with the individual's cancer cells is determined and mutations corresponding with the cancer are identified.

A guide RNA comprising a sequence from Table 6 and paragraph [00303] is then synthesized and is assembled into a ribonucleoprotein complex with a polypeptide comprising Cas12a nuclease domain. Alternatively, or in combination, a guide RNA comprising a sequence from Table 7 and paragraph [00305] is synthesized and is assembled into a ribonucleoprotein complex with a polypeptide comprising a Cas9 nuclease domain. The complex, comprising Cas12a, Cas9, or both, are then administered to the individual and symptom amelioration and tumor size reduction is observed.

Example 13. Small Molecule Combination Therapy

The individual of any of the Examples herein is additionally administered a small molecule inhibitor of DNA double strand break repair to enhance genome editing efficiency. The inhibitor can target a gene involved in double strand break repair, for example, associated with BRCA pathway or BRCA gene. Upon administration, symptom amelioration and disease remission is observed in the individual.

Example 14. Macromolecule Combination Therapy

The individual of any of the Examples herein is additionally administered a macromolecular inhibitor of DNA double strand break repair to enhance genome editing efficiency. A guide RNA is designed to target a gene involved in double strand break repair, for example, associated with BRCA pathway or BRCA gene. The guide RNA is administered to the individual along with a Cas nuclease such as Cas9 or Cas12a. Symptom amelioration and lung cancer remission is observed in the individual upon administration of the combination therapy.

Example 15. Treatment of Adenocarcinoma with PX459 Plasmid DNA

A Cas9 expression vector (PX459, Addgene plasmid #62988) with mutant specific guide RNA was used to induce multi-cleavage in EGFR mutant lung cancer genome. HCC827, a lung adenocarcinoma cell line, was electroporated for the delivery of the Cas9/sgRNA expression vector. HCC827 has E2 mutation in its EGFR gene and there are more than 18 copies of the mutation sequence in the specific genome. The guide RNA was designed so that it targets more than 18 loci in the HCC827 cell line. Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they fill up around 50% of the area, trypsinized, washed with PBS, and resuspended in Neon Electroporation Buffer R. 150,000 cells with 500 ng of the Cas9/sgRNA expression were loaded in a 10 μL Neon Pipette tip, and electroporated under optimized conditions (1300V, 20 ms, 2 pulses). Cells were recovered in the growth media for six days and counted. FIG. 6 shows Cas9 guide RNA derived multi-cleavage induction and cell death in an EGFR mutant lung cancer cell line, HCC827, six days after neon electroporation of PX459 plasmid DNA targeting CNV. Compared to the control groups that were electroporated without any vectors or with vectors that target the wild-type EGFR sequence, the experimental group electroporated with vectors that target the HCC827 specific mutation had a significantly lower number of cells. The results additionally showed that the experimental group (EGFR_E2) showed 83% cell death. The results showed that multi-cleavage induction in adenocarcinoma by Cas9 and target specific guide RNA reduced cell proliferation.

Example 16. H1299 Transfection without Drug Treatment

H1299 cells were plated at 1.5×10⁵ cells per well in a 24-well plate, and 500 ng of plasmids were introduced into each wells as described above in TABLE 8 below.

TABLE 8 NT1 CCR5 HPRT1 MT2 SMIM11 GNPDA2 SLC15A5 KCNE2 Cut number 0 2 2 >100 >40 >12 >12 >40 essentiality N/A Non House Non Non oncogene oncogene Non essential keeping essential essential essential

Transfection was performed 24 hours after from cell plating, by using Lipofectamine3000 reagent according to the protocol served by manufacturer. 72 hours post-transfection, all supernatant was discarded by suction, and cells were washed gently with 500 μL of 1×PBS. Cells were harvested by trypsinization, and the live cell number was counted with trypan blue dye and cell countess. CCR5, HPRT1, the targets only making two cuts in the whole genome, and NT1, having no matched sequence in the human genome, showed similar cell viability. In contrast, MT2, known to make multiple cuts at more than 100 loci, showed about 50% cell viability relative to NT1. H1299 cell line specifically amplified loci, SMIM11, GNPDA2, SLC15A5, and KCNE2, showed similar cell viability as MT2, while SMIM11 and KCNE2 showed higher induction of cell death, having about 25% of cell viability relative to NT1.

Example 17. H1299 Transfection without Drug Treatment-2

H1299 cells were plated as 1.5×10{circumflex over ( )}5 cells per well in a 24-well plate, and 500 ng of plasmids were introduced into each wells as described below in TABLE 9.

TABLE 9 Lipo only NT1 CCR5 HPRT1 MT2 SMIM11 GNPDA2 SLC15A5 KCNE2 cut N/A 0 2 2 >100 >40 >12 >12 >40 number essentiality N/A N/A Non- House- Non- Non- oncogene oncogene Non- essential keeping essential essential essential

Transfection was performed at 24 hours after cell plating, by using Lipofectamine3000 reagent according to the protocol served by manufacturer. 48 hours post-transfection, all supernatant was discarded by suction, and cells were washed gently with 500 μL of 1×PBS. Cells were harvested by trypsinization, and live cell number was counted with trypan blue dye and cell countess. CCR5, targets making two cuts in whole genome, showed 80% cell viability relative to lipofection only control. HPRT1, making two cuts in a house keeping gene, showed more toxicity with about 50% cells alive. NT1, having no matched sequence in the human genome, showed 57% cell viability compared with the lipofection only. Whereas, MT2, having multiple cut sites at more than 100 loci, showed about 14% cell viability relative to lipofection only control. The H1299 cell line specifically amplified locus targets, GNPDA2, SLC15A5, KCNE2, showed total cell death that was not reliably counted, while showed 57% cell viability compared with lipofection only. Two samples (NT1, GNPDA2) were also imaged by using a NucBlue live cell stain reagent (Blue, staining live and dead cells) and Propidium iodide dead cell stain reagent (Red, only staining the dead cells), that showed consistent result in FIG. 10.

Example 18—H1563 Transfection

H1563 cells were plated at 1.5×10⁵ cells per well in a 24-well plate, and 500 ng of plasmids were introduced into each wells as described below in TABLE 10.

TABLE 10 CCR5 HPRT1 MT2 IRX1 ADAMTS16 cut number 2 2 >100 >8 >7 essentiality Non House Non Non Non essential keeping essential essential essential

Transfection was performed 24 hours after cell plating, by using Lipofectamine3000 reagent according to the protocol served by manufacturer. 24 hours post-transfection, cells were treated with 1 μg/mL of puromycin and cells were selected for 72 hours. All supernatant was discarded by suction, and cells were washed gently by 500 μL of 1×PBS. Cells were harvested by trypsinization, and live cell number was counted with trypan blue dye and cell countess. CCR5, a target with two cuts in the whole genome, showed the highest cell viability and HPRT1 showed 52% of it. Targets making multiple cuts (MT2, IRX1, and ADAMTS16) showed similar cell viability to each other. MT2 showed 43% cell viability relative to CCR5, and H1563 line specific amplified targets showed much more toxicity. ADAMTS16 resulted in 40% cell viability and IRX1 resulted in 37% cells that were alive compared to CCR5.

Example 19: Cell Death of A549 Cells by Multi-Target 2

FIG. 12-FIG. 13 shows cell death of A549 cells by multi-target 2. 500 ng of Cas9 protein and gRNA-expressing DNA were introduced into A549 (pancreatic) cells by electroporation. The cultured A549 cells were washed with 1×PBS, and removed from the floor using trypsin-EDTA. The required number of cells was removed and washed once with 1×PBS. Cells were resuspended with Lonza SF buffer at 20 μL/each for each condition and mixed with each DNA. The cell and DNA mixture was transferred to a Lonza electroporator and electric shocked. As a control, NT1 (non target), which is not aligned in the human genome, HPRT1, which is a house keeping gene, and CCR5, which has 1 copy, and electric shock only condition were added. After introducing DNA by electric shock, cells were plated into 24 wells in duplicate and 96 wells in 3 replicates. After 24, 42 and 72 hours of DNA introduction, 50 μL of CellTiter Glo reagent (Promega, G9241) was added to the 96 well plate. Plates were placed on a FLUOstar omega reader machine and shaken for 2 minutes and allowed to react for 10 minutes at room temperature. Afterwards, the degree of luminescence was measured. This method is based on the amount of ATP in the cell, and the amount of living cells undergoing metabolism is determined by absorbance. As compared to the three control conditions, the MT2 (multitarget 2) condition targeting over 100 sites in the human genome, 20˜50% cell death was induced over time. In addition, three CNV targets of CD68, DACH2, and HERC2P2 induced cell death similar to MT2 and SHBG2 CNV target induced 70˜80% cell death compared to control condition. After 24 hours of DNA introduction, 1 μg/mL of puromycin was added to the 24 well, and the cells were recovered by changing the cell culture medium at the time of almost cell death. After recovery for 5˜7 days, cells were removed from each well and stained with trypan blue to determine the number of viable cells. After the DNA was introduced, the cells were selected with puromycin and then observed under a microscope before measuring the number of cells. Similar to the results of cell counting, in HPRT1, CCR5 targeted treatments, more than 50% of the cells were recovered, whereas 90% cell death was observed in MT2 targeting treatments and only 10% of the cells was recovered. Therefore, it was confirmed that the cell death was induced when the CNV target DNA break was induced using the Cas9 protein in A549 cells.

Example 20: Electroporation Lonza: A549 (CellTiter Glo), Cell Death of A549 Cells by CNV Target

A549 (pancreatic) cells were transfected with 500 ng of Cas9 protein and DNA encoding each CNV target gRNA by electroporation. The cultured A549 cells were washed with 1×PBS, and removed from the floor using trypsin-EDTA. The required number of cells was removed, washed once with 1×PBS. Cells were resuspended with Lonza SF buffer at 20 μL/each condition, and mixed with each DNA. The cell and DNA mixture was transferred to a Lonza electroporator and subject to electric shock. As controls, a condition in which a pET21a vector capable of expressing a protein in E. coli, an electric shock only condition, and no treatment were added. After introducing DNA by electric shock, the cells were added to the 96 wells in 3 replicates per each condition. After 24, 44, and 51 hours of DNA introduction, 50 μL of CellTiter Glo reagent was added to each well. Plates were placed in a FLUOstar omega reader and shaken for 2 minutes. After reacting at room temperature for 10 minutes, the luminescence was measured. This method involves metabolism based on the amount of ATP in the cell Is the method of determining the degree of living cells by absorbance. As a result, 20˜50% cell death was induced over time in MT2 (multitarget 2) conditions, targeting over 100 sites in human genome, compared with the three control conditions. In addition, the three CNV targets of CD68, DACH2, and HERC2P2 observed a similar degree of cell death to the MT2 condition, and SHBG CNV target induced 70˜80% cell death compared to control conditions. Therefore, it was confirmed that the cell death was induced when the CNV target DNA break was induced using the Cas9 protein in A549 cells.

Example 21: Cell Death of SKBR3 Cells by CNV Target

SKBR3, a breast cancer cell line, was treated with 500 ng of Cas9 protein and DNA expressing each CNV target gRNA by electroporation. The cultured SKBR3 cells were washed with 1×PBS, and then treated with trypsin-EDTA and removed from the bottom. The required number of cells was removed, washed once with 1×PBS. Cells were resuspended with Lonza SF buffer 20 μL/each condition, and mixed with each DNA. The cell and DNA mixture was placed in a Lonza electroporator and subjected to electrical shock. NT1 (non target), not aligned in the human genome, electric shock only condition, and no treatment condition were added as control conditions. After introducing DNA by electric shock, cells were inserted into the 96 wells in 3 replicates per each condition. 24, 42 and 48 hours after DNA introduction, 50 μL of CellTiter Glo reagent was added to 96 wells. Plates were placed in a FLUOstar omega reader and shaken for 2 minutes. After reacting at room temperature for 10 minutes, the luminescence was measured. This method was based on the amount of ATP in the cell, and the amount of living cells undergoing metabolism was determined by absorbance. As a result, 30% cell death was induced in the MT2 condition targeting over 100 sites in human genome, compared to three control conditions. In addition, two CNV targets, ERBB2 and KRT1,6 induced 40˜50% cell death compared to control conditions. After introducing DNA by electric shock, the cells were put into 24 wells in duplicate. 48 hours after the introduction of the DNA, the cells were removed from each well of 24 wells and stained with trypan blue to measure the number of living cells. As a result, 40% cell death was induced in MT2 condition compared to control condition, and 2 types of ERBB2 and KRT16 CNV targets induced 40˜50% cell death compared with control condition. Therefore, it was confirmed that cell death was induced by inducing target DNA break using Cas9 protein in two CNVs of SKBR3 cells.

Example 22: Targeting the HeLa CNV or HPV Gene for Treatment of Cervical Cancer

Shown in FIG. 18A is an HPV duplicated loci map in the HeLa genome from Adey, A. et al. (The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500, 207-211 (2013)). Purple indicates HeLa and green indicates HPV. Shown in FIG. 18B is HPV sequence regions in a HeLa cell, inferred from Meissner et al. (Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J. Gen. Virol. 80, 1725-1733 (1999)), Landry et al. (The genomic and transcriptomic landscape of a HeLa cell line. G3:Genes|Genomes|Genetics 3, 1213-24 (2013)), Adey, and Liu et al. (Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology. Oncotarget 7, 5852-5864 (2016)). Repeat numbers are marked as X and only opened sequences are marked as arrow. Shown in FIG. 18C is an electrophoresis gel of PCR product which indicate the repeat region between HPV sequences in a HeLa cell, based on Meissner, Landry, Adey, and Liu.

600 ng of a DNA vector expressing Cas9 and gRNA was electroporated in 2×10⁵ cells of a cervical cancer model cell (HeLa cell). To prepare HeLa cells, media was removed from the flask, cells were washed with 1×PBS, and cells were treated with Trypsin-EDTA to detach the HeLa cells from the flask. Cell counting was performed, the appropriate number of cells was transferred to a new tube, cells were washed with 1×PBS, and cells were resuspended with Lonza SE buffer and mixed with the DNA vector. The mixture was transferred to an eletroporator and electroporation was carried out. A plasmid targeting a non essential gene, CCR5, whose copy number is one, was used as a negative control, and a mixture of plasmids targeting 100 non essential genes (MT2) was used as positive control. In the experimental group, plasmid targeting the non essential gene PRDM9 duplicated 8 times and the human papillomavirus (HPV) gene duplicated 30 times in HeLa cell were targeted. 24 hours after introducing DNA by electroporation, the media was removed and replaced with 0.5 ug/ml puromycin to select transfected HeLa cell. 3 days after puromycin selection, media was replaced with standard media, cells were cultivated for 3-4 days, and cells were counted. FIG. 19 shows graphs of cell viability for each group. The data demonstrated that targeting MT2 induces cell death up to 50% as compared to the negative control, CCR5. In addition, targeting PRDM9 and HPV_1 sequences induced cell death similar to or more than targeting MT2. Therefore, Hela cell specific CNV & HPV genes were cut by Cas9 and cell death was induced.

FIG. 20 was generated using the same methods described above. 24, 48, and 72 hours post-electroporation, the amount of living cells undergoing metabolism was measured using Cell Titer Glo 2.0, which quantitates ATP by measuring luminescence absorbance. A GFP expressing vector (GFP) was used and the non essential gene CCR5, whose copy number is one, was used as a negative control. A mixture of plasmid targeting 100 non essential genes (MT2) was used as a positive control. In experimental groups, plasmids targeted the non essential gene PRDM9, duplicated 8 times, and the human papillomavirus (HPV) gene, duplicated 30 times in a HeLa cell. FIG. 20A shows graphs of the luminescence signal, or ATP level, for each treatment group at each time point. The data demonstrated that in HeLa cells, there is a similar luminescence signal between pulse only (GFP) and CCR5. Targeting MT2 induced cell death up to 50% as compared to the negative control, CCR5. In addition, targeting PRDM9 and HPV_1 sequences induced cell death similar to or more than the MT2 condition. HeLa cell death after targeting the HPV gene was assessed by a cell titer glo assay, as follows. FIG. 20B was generated using the same methods described above. A plasmid was used to target a non essential gene, CCR5, whose copy number is one as negative control, and a mixture of plasmids targeting 100 non essential gene (MT2) was used as positive control. A non-target negative control (NT1) was added, which targets non-human sequence. The NT1 control has a 20mer spacer, and NT2/NT3 have 10mer, 5mer spacer each. As the experimental group, a plasmid targeting human papillomavirus (HPV) gene was used, which is duplicated 30 times in HeLa cell. 24 hours after introducing DNA by using electroporation, media was removed and replaced with 0.5 ug/ml puromycin to select for transfected HeLa cell. 3 days after puromycin selection, media was replaced with standard media, cultivated for 10 days. The relative amount of living cells undergoing metabolism was calculated by Cell titer Glo. As a result of experiments, in HeLa cell, CCR5 showed 75% luminescence signal than NT3 whose spacer sequence are 5mer. Also, targeting MT2 induced cell death up to 99.5% than NT3. In addition, targeting HPV_1 sequence induced cell death up to about 90% than CCR5 condition. Therefore, when Hela cell specific HPV gene was cut by Cas9, cell death was induced, as detected by a Cell Titer Glo.

Example 23: HT29 Transfection Targeting Colon Cancer Relevant Genes

HT29 cells were plated at 1.5×10⁵ cells per well in a 24-well plate, and 500 ng of plasmids were introduced into each wells as described in TABLE 11.

TABLE 11 Lipo only GFP NT1 CCR5 HPRT1 MT2 TRAPPC9 LINC00536 TRPS1 CDK8 cut N/A 0 2 2 2 >100 >13 >9 >8 >18 number essentiality N/A N/A N/A Non- House- Non- Non- Non- Non- Oncogene essential keeping essential essential essential essential

Transfection was performed at time point 24 hours after from cell plating, by using Lipofectamine3000 reagent according to manufacturer's protocol. 24 hours post-transfection, cells were treated with 1 μg/mL of puromycin and cells were selected at 90 hours. Almost total cell death was observed because of the low efficiency of transfection, and a few survived cells were recovered by normal complete McCOY media at 12 days. All supernatant was discarded by suction, and cells were washed gently by 500 μL of 1×PBS. Cells were harvested by trypsinization, and live cell number was counted with trypan blue dye and cell countess. FIG. 21 shows the live cell count per treatment group. CCR5, the target only making two cuts in whole genome, and NT1, having no matched sequence in a human genome, showed similar high levels of cell viability. Whereas, HPRT1, making two cuts in house keeping gene showed about 50% cell viability compared to NT1. Multiple cut making targets showed heavy cell death, especially MT2, TRAPPC9, and CDK8, which showed little or no recovered cells. LINK00536 and TRPS1 showed 18% and 20% cell viability compared to NT1 respectively.

Example 24: Target Cross Check (1299 Targets in 1563)

FIG. 22 was performed alongside the H1563 transfection shown in FIG. 13. H1563 cells were plated as 1.5×10⁵ cells per well in a 24-well plate, and 500 ng of plasmids were introduced into each wells as described above in TABLE 12 below.

TABLE 12 CCR5 SMIM11 GNPDA2 SLC15A5 KCNE2 cut number 2 2 2 2 2 essentiality Non Non oncogene oncogene Non essential essential essential

Transfection was performed 24 hours after cell plating, by using Lipofectamine3000 reagent according to the manufacturer's protocol. 24 hours post-transfection, cells were treated with 1 μg/mL of puromycin and selected at 72 hours. All supernatant was discarded by suction, and cells were washed gently by 500 μL of 1×PBS. Cells were harvested by trypsinization, and the live cell number was counted with trypan blue dye and cell countess. FIG. 22 shows a graph of live cells in each treatment group. H1299 line specific CNV targets SMIM11, GNPDA2, SLC15A5, KCNE2 were tested in H1563, where they only make two cuts in the whole genome. As expected, SMIM11, GNPDA2, SLC15A5 showed no harmful effects compared to CCR5. KCNE2 showed cell death effect. This phenomenon was also confirmed in microscopic images (FIG. 23).

Example 25: Lipofection in H1299 Cells, Dead Cell Determination by Annexin V Positive Determinations

One day before transfection, H1299 cells, a lung cancer cell line, were plated in a white 96-well plate at 1.3×10⁴. On the next day, 500 ng of Cas9 protein and DNA encoding each CNV target gRNA was introduced using liposomes. 0.3 μL of liposome reagent I was mixed with 5 μL of opti-MEM to make tube 1. 0.2 μL of liposome reagent II was mixed with 5 μL of opti-MEM and 500 ng of DNA for each experimental condition to make tube 2. Tube 2 was mixed with tube 1 and incubated at room temperature for 15 minutes. 11 μL of liposome and DNA mixture was added to 96 wells. After 3 hours, AnnV reagent as added and the degree of luminescence was measured 24 hours after lipofection. This method measures the extent of apoptosis according to the degree of luminescence when AnnV is added to the PS (phosphatidylserine) site exposed to the outer cell membrane when apoptosis occurs. As a result, when the target DNA break was induced by using Cas9 protein in the four CNVs of H1299 cells, and 30% to 40% cell death rate was observed. The cell membrane was exposed to PS to induce apoptosis. FIG. 24 shows a graph of luminescence, indicating cell death, for each treatment group.

Example 26: Cas9 Guide RNA Derived Multi-Cleavage Induction and Cell Death in EGFR Mutant Lung Cancer Cell Line, HCC827, and Enhancing Cell Killing Effect by Human Codon-Optimized Rec J Proteins

A Cas9 expression vector (PX459, Addgene plasmid #62988) with mutant specific guide RNA was used to induce multi-cleavage in an EGFR mutant lung cancer genome. A vector expressing Cas9-RecJ protein was cloned, wherein the Cas9 sequence was flanked by human codon-optimized Rec J sequence at the 3′ end. HCC827, a lung adenocarcinoma cell line, was electroporated for delivery of the Cas9/sgRNA expression vector. HCC827 cells have an E2 mutation in their EGFR gene and there are more than 18 copies of the mutation sequence in the specific genome. The guide RNA was designed so that it targets more than 18 loci in HCC827 cell line. Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they filled around 50% of the area, trypsinized, washed with PBS, and finally resuspended in Neon Electroporation Buffer R. 150,000 cells with 500 ng of the Cas9/sgRNA expression were loaded in a 10 μL Neon Pipette tip, and electroporated with an optimized condition (1300V, 20 ms, 2 pulses). Cells were recovered in the growth media for 4 days and counted. FIG. 25, at bottom, shows a graph of live cells in each treatment group. Compared to the control groups that were electroporated without any vectors, with vectors that target the wild-type EGFR sequence, or with a vector targeting a single locus (CCR5), the experimental group that was electroporated with vectors that target the HCC827 specific mutation had significantly lower number of cells. 33% cell death was observed in the EGFR_E2 experimental group. Moreover, it was confirmed that the Cas9RecJ protein not only enhanced the multi-cleavage effect, but it also conferred cell killing capability to single loci targeting guide RNAs. The results showed that multi-cleavage induction in an adenocarcinoma by Cas9 and target specific guide RNA reduced cell proliferation, and the effects were enhanced with human codon-optimized Rec J proteins. 50% cell death was observed in the CCR5 group and 80% cell death was observed in the EGFR_E2 group. Using the Cas9-RecJ constructs increased the cell death efficiency by 5-fold as compared to the control.

Example 26: Various Delivery Methods

This example illustrates various delivery methods to administer CRISPR-Cas9 constructs.

RNP neon electroporation in HCC827 cells. FIG. 26 shows Cas9 guide RNA derived multi-cleavage induction and cell death in an EGFR mutant lung cancer cell line, HCC827. A Cas9/sgRNA ribonucleoprotein (Cas9 RNP) with mutant specific guide RNA was used to induce multi-cleavage in the EGFR mutant lung cancer genome. HCC827, a lung adenocarcinoma cell line, was electroporated for delivery of the Cas9 RNPs. HCC827 has E2 mutation in its EGFR gene and there are more than 18 copies of the mutation sequence in the specific genome. The guide RNA was designed to target more than 18 loci in HCC827 cell line. Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they fill up around 50% of the area, trypsinized, washed with PBS, and finally resuspended in Neon Electroporation Buffer R. 150,000 cells with 1.2 μM of RNPs were loaded in a 10 μL Neon Pipette tip, and electroporated with an optimized condition (1300V, 20 ms, 2 pulses). Cells were recovered in the growth media for 24 hours and counted. Compared to the control groups that was electroporated without any complete RNPs, the experimental group electroporated with RNPs targeting the HCC827 specific mutation had significantly lower number of cells by 3-fold as compared to the non-targeted RNP. The results showed that multi-cleavage induction in a adenocarcinoma by Cas9 and target specific guide RNA reduced cell proliferation.

Lipofection of Cas9 Constructs in 111563 cells. FIG. 27 shows Cas9 guide RNA derived multi-cleavage induction and cell death in an adenocarcinoma cell line, H1563. A Cas9/sgRNA ribonucleoprotein (Cas9 RNP) with a guide RNA was used to target more than 100 loci in human genome. H1563, a lung adenocarcinoma cell line, was electroporated for the delivery of the Cas9 RNPs. Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they fill up around 50% of the area, trypsinized, washed with PBS, and finally resuspended in Neon Electroporation Buffer R. 150,000 cells with 1.2 μM of RNPs were loaded in a 10 μL Neon Pipette tip, and electroporated with an optimized condition (1200V, 20 ms, 2 pulses). Cells were than recovered in the growth media for 48 hours and counted. Compared to the control groups that was electroporated with non-targeted RNPs, the experimental group that was electroporated with RNPs that target the multiple loci had significantly lower number of cells by 30%. The results showed that multi-cleavage induction in a adenocarcinoma by Cas9 and target specific guide RNA reduced cell proliferation.

H1299 RNP (Cas9-GFP) 2 days after Neon electroporation. FIG. 28 shows Cas9 guide RNA derived multi-cleavage induction and cell death in an adenocarcinoma cell line, H1299. Cas9/sgRNA ribonucleoproteins (Cas9 RNPs) with mutant specific guide RNAs was used to induce multi-cleavage in the H1299 lung cancer cell line. Genes with high copy-number variation (CNV) in H1299 genome were searched and GNPDA2 (>12 copies, oncogene) and SMIM11 (>40 copies, non-essential gene) were selected as Cas9 targets. A guide RNA (MT2) was designed that targets more than 100 loci in human genome and used it for the experiment. H1299, a lung adenocarcinoma cell line, was electroporated for the delivery of the Cas9 RNPs. GFP-tagged Cas9 proteins were used to verify the transfection. Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they fill up around 50% of the area, trypsinized, washed with PBS, and finally resuspended in Neon Electroporation Buffer R. 150,000 cells with 1.2 μM of RNPs were loaded in a 10 μL Neon Pipette tip, and electroporated with an optimized condition (1300V, 20 ms, 2 pulses). Cells were than recovered in the growth media for 48 hours and counted. Compared to the control groups that was electroporated without any complete RNPs, the experimental groups that were electroporated with RNPs that target multiple loci in the H1299 genome had significantly lower numbers of cells. 71% cell death was observed in the GNPDA2 experimental group, 40% cell death was observed in the SMIM11 experimental group, and 33% cell death was observed in the MT2 experimental group. The results showed that multi-cleavage induction in a adenocarcinoma by Cas9 and target specific guide RNA reduced cell proliferation.

Example 27: Cell Death of HT-29 Cells by Targeting CNVs

To assay specific killing in HT-29 (colon cancer cell line) by targeting CNV and MT2. HT-29 cells were introduced with 500 ng of Cas9 protein and DNA expressing each CNV target gRNA by electroporation. The cultured HT-29 cells were washed with 1×PBS, and then treated with trypsin-EDTA and removed from the bottom. The required number of cells was removed and washed once with 1×PBS. Cells were resuspend with Lonza SF buffer 20 μL/each condition, and mixed with each DNA. The cell and DNA mixture was placed in a Lonza electroporator and subjected to electrical shock. NT1 (non target), which is not aligned in the human genome and electric shock only condition were added as control conditions. After introducing DNA by electric shock, cells were inserted into the 96 wells in 4 replicates per each condition. After 24 hours of DNA introduction, 50 μL of CellTiter Glo reagent was added to 96 wells. Plates were placed in a FLUOstar omega reader and shaken for 2 minutes. After reacting at room temperature for 10 minutes, the luminescence was measured. This method is based on the amount of ATP in the cell, and the amount of living cells undergoing metabolism is determined by absorbance. As a result, 90% cell death was induced in the TRAPPC9 and MT2 condition targeting over 100 sites in human genome, compared to NT1 conditions. In addition, three CNV targets, CDK8, LINC00536 and TRPS1 induced 20˜45% cell death, as shown in FIG. 29.

Example 28: Sequence Specific Cancer Cell Killing Effect by Cas12a and Single or Multi-Target crRNAs

A Cas12a expression vector was used with crRNAs that target a high CNV mutation sequence (EGFR_E2) or a non-CNV WT sequence (CCR5). The crRNAs were designed to induce either >18 cleavages or a double cleavage in HCC827 genome, respectively. HCC827, a lung adenocarcinoma cell line, was electroporated for the delivery of the Cas12a/crRNA expression vector. HCC827 has the E2 mutation in its EGFR gene and there are more than 18 copies of the mutation sequence in the specific genome. The Cas12a DNA sequence used was ATGGGCAAAAACCAAAATTTCCAAGAATTTATCGGAGTGAGCCCCCTGCAGAAaACCCTC CGGAACGAGCTTATTCCGACTGAGACCACAAAGAAAAATATAACCCAGCTGGACTTGCTG ACTGAAGATGAGATCCGCGCCCAGAACCGGGAAAAGCTCAAAGAGATGATGGACGATTA TTACCGCAATGTTATTGACAGTACCCTTCACGTCGGGATCGCTGTGGATTGGTCTTATCTG TTCAGCTGCATGCGGAACCATTTGCGCGAAAATTCCAAGGAGTCAAAACGGGAACTGGAG CGCACACAGGACAGCATTCGGAGTCAGATACACAACAAGTTTGCCGAACGCGCAGATTTC AAAGACATGTTTGGCGCCTCTATCATTACCAAGCTCCTTCCTACTTACATCAAACAAAATA GCGAGTATTCCGAACGGTACGATGAGTCAATGGAAATTCTGAAGTTGTATGGTAAATTCA CCACAAGCCTGACCGACTACTTTGAGACTCGCAAGAACATATTCAGTAAAGAAAAGATCT CTAGCGCTGTAGGCTATCGGATTGTGGAGGAAAATGCCGAGATCTTTCTCCAGAACCAGA ATGCATACGATCGCATTTGTAAAATAGCCGGACTTGACCTGCATGGGTTGGATAACGAAA TCACCGCTTATGTTGACGGCAAGACACTGAAAGAGGTCTGCTCCGATGAAGGTTTCGCCA AGGCAATTACCCAAGAGGGCATCGAtCGGTACAATGAAGCCATTGGAGCTGTGAACCAGT ATATGAATCTCCTTTGTCAGAAAAACAAGGCCCTGAAACCCGGGCAATTTAAGATGAAAC GCTTGCACAAGCAGATACTGTGCAAAGGCACTACCTCATTCGATATCCCGAAGAAATTTG AGAATGACAAGCAGGTATACGATGCAGTGAACAGCTTCACAGAAATTGTTACCAAAAAT AACGACCTCAAGCGGCTTCTGAATATCACTCAAAACGCCAATGATTATGACATGAACAAA ATTTACGTCGTGGCTGATGCCTATAGTATGATATCTCAGTTTATCAGCAAGAAATGGAATT TGATTGAGGAATGTCTGCTCGACTACTATTCCGATAACCTTCCAGGTAAGGGCAATGCAA AAGAGAACAAGGTAAAAAAGGCCGTGAAAGAAGAGACCTACCGCTCAGTTAGCCAGCTG AATGAAGTCATCGAGAAGTATTACGTGGAAAAAACAGGACAAAGTGTATGGAAGGTGGA GTCTTATATTAGCTCCTTGGCTGAAATGATAAAACTGGAGCTCTGCCATGAAATCGACAA CGATGAGAAGCACAATCTTATTGAgGACGATGAGAAAATCTCAGAAATTAAGGAGCTGTT GGACATGTACATGGATGTTTTCCATATAATCAAAGTCTTTCGGGTGAACGAAGTACTGAA TTTCGACGAGACCTTTTATAGCGAAATGGATGAGATTTACCAGGACATGCAGGAAATCGT GCCCCTCTATAACCACGTTCGCAATTACGTCACTCAAAAGCCGTATAAACAGGAGAAGTA CCGGCTTTATTTCCATACCCCTACACTGGCCAACGGGTGGAGTAAATCTAAGGAATACGA TAATAACGCAATTATATTGGTGCGCGAGGACAAATATTACCTGGGCATCCTCAATGCCAA GAAAAAGCCCAGCAAAGAAATTATGGCTGGTAAGGAGGATTGTTCCGAACACGCCTATG CAAAAATGAACTACTATCTTCTGCCGGGCGCCAATAAGATGTTGCCAAAAGTATTTCTGTC AAAGAAAGGAATCCAGGACTACCATCCCAGCAGTTATATTGTGGAGGGGTACAACGAAA AGAAACACATAAAGGGCTCTAAAAATTTCGATATCCGGTTTTGCCGCGACCTCATTGATT ATTTCAAGGAGTGTATCAAAAAGCATCCGGACTGGAACAAATTTAATTTCGAATTTAGCG CTACCGAGACTTACGAAGATATTTCCGTTTTCTATCGGGAGGTCGAAAAGCAAGGTTACC GCGTGGAGTGGACCTATATAAACTCAGAgGACATCCAGAAACTTGAGGAAGATGGCCAGC TGTTTTTGTTCCAAATTTACAATAAGGACTTTGCCGTAGGAAGCACAGGGAAACCTAACCT GCACACCCTCTATCTTAAGAATCTGTTCAGTGAGGAAAACTTGCGGGATATCGTGCTGAA ACTCAATGGCGAGGCAGAAATTTTTTTCCGCAAGTCTAGCGTTCAGAAACCCGTCATACA TAAGTGCGGTTCCATCCTTGTGAACCGGACTTACGAGATTACCGAATCAGGCACAACCCG CGTACAGAGCATCCCGGAGAGTGAATATATGGAGCTGTAtCGGTATTTTAATTCTGAAAAA CAAATTGAGTTGAGCGACGAAGCCAAGAAATACCTGGATAAGGTGCAGTGTAACAAAGC TAAGACTGACATAGTTAAAGATTATCGCTACACCATGGACAAGTTCTTTATCCACCTCCCA ATTACAATCAATTTCAAAGTCGATAAGGGAAACAATGTGAACGCCATTGCACAGCAATAT ATAGCCGGGCGGAAAGACCTTCATGTAATCGGCATTGATCGCGGTGAGCGGAATCTGATC TACGTGTCCGTTATTGACATGTATGGCCGCATATTGGAACAGAAGTCATTTAACCTGGTCG AGCAGGTGAGCAGTCAAGGAACCAAACGGTACTATGATTACAAGGAAAAACTCCAGAAT CGCGAGGAAGAGCGGGACAAGGCTCGCCAGTCTTGGAAAACTATCGGGAAGATTAAAGA ACTTAAGGAGGGCTATCTGAGCTCCGTAATCCACGAAATTGCCCAAATGGTGGTTAAATA CAACGCAATAATCGCCATGGAGGATTTGAATTATGGTTTCAAGCGGGGCCGCTTTAAAGT CGAACGGCAGGTGTACCAGAAGTTCGAGACCATGCTGATTTCAAAACTCAACTATCTTGC TGACAAGAGCCAAGCCGTAGATGAACCCGGAGGGATTCTGCGCGGCTACCAGATGACAT ATGTGCCGGACAATATTAAAAACGTTGGTCGGCAGTGCGGCATAATCTTTTACGTCCCTGC AGCCTATACCAGTAAGATTGATCCCACTACCGGATTCATCAATGCTTTTAAACGCGACGTG GTATCTACAAACGATGCCAAGGAGAATTTCTTGATGAAATTTGACAGCATTCAATACGAT ATAGAAAAGGGGCTGTTCAAATTTTCCTTCGACTATAAGAACTTTGCAACCCATAAACTC ACTCTTGCCAAGACCAAATGGGATGTGTACACAAATGGCACCCGGATTCAGAACATGAAG GTTGAGGGTCACTGGCTGTCAATGGAAGTCGAGTTGACTACCAAAATGAAGGAACTGCTC GACGATAGCCATATTCCGTATGAGGAAGGCCAGAATATCCTTGACGATCTGCGCGAGATG AAAGACATTACAACCATAGTGAACGGAATCTTGGAAATTTTCTGGCTGACTGTACAACTC CGGAATAGTCGCATCGATAACCCAGACTACGATCGGATTATATCTCCCGTGCTTAATAAG AACGGGGAGTTTTTCGACAGCGATGAATATAATTCCTACATCGACGCTCAGAAAGCCCCG CTGCCTATTGATGCAGACGCCAACGGCGCTTTTTGTATCGCCTTGAAGGGTATGTATACCG CAAATCAGATTAAAGAGAACTGGGTTGAAGGCGAGAAGCTGCCCGCCGATTGCCTCAAA ATAGAACACGCTTCATGGCTTGCCTTCATGCAAGGAGAGCGCGGG (SEQ ID NO: 3286) and the corresponding protein sequence was MGKNQNFQEFIGVSPLQKTLRNELIPTETTKKNITQLDLLTEDEIRAQNREKLKEMMDDYYRN VIDSTLHVGIAVDWSYLFSCMRNHLRENSKESKRELERTQDSIRSQIHNKFAERADFKDMFGA SIITKLLPTYIKQNSEYSERYDESMEILKLYGKFTTSLTDYFETRKNIFSKEKISSAVGYRIVEEN AEIFLQNQNAYDRICKIAGLDLHGLDNEITAYVDGKTLKEVC SDEGFAKAITQEGIDRYNEAIG AVNQYMNLLCQKNKALKPGQFKMKRLHKQILCKGTTSFDIPKKFENDKQVYDAVNSFTEIVT KNNDLKRLLNITQNANDYDMNKIYVVADAYSMISQFISKKWNLIEECLLDYYSDNLPGKGNA KENKVKKAVKEETYRSVSQLNEVIEKYYVEKTGQSVWKVESYISSLAEMIKLELCHEIDNDEK HNLIEDDEKISEIKELLDMYMDVFHIIKVFRVNEVLNFDETFYSEMDEIYQDMQEIVPLYNHVR NYVTQKPYKQEKYRLYFHTPTLANGWSKSKEYDNNAIILVREDKYYLGILNAKKKP SKEIMA GKEDCSEHAYAKMNYYLLPGANKMLPKVFLSKKGIQDYHPSSYIVEGYNEKKHIKGSKNFDI RFCRDLIDYFKECIKKHPDWNKFNFEFSATETYEDISVFYREVEKQGYRVEWTYINSEDIQKLE EDGQLFLFQIYNKDFAVGSTGKPNLHTLYLKNLFSEENLRDIVLKLNGEAEIFFRKSSVQKPVI HKCGSILVNRTYEITESGTTRVQSIPESEYMELYRYFNSEKQIELSDEAKKYLDKVQCNKAKTD IVKDYRYTMDKFFIELPITINFKVDKGNNVNAIAQQYIAGRKDLHVIGIDRGERNLIYVSVIDM YGRILEQKSFNLVEQVSSQGTKRYYDYKEKLQNREEERDKARQSWKTIGKIKELKEGYLSSVI HEIAQMVVKYNAIIAMEDLNYGEKRGREKVERQVYQKFETMLISKLNYLADKSQAVDEPGGI LRGYQMTYVPDNIKNVGRQCGIIFYVPAAYTSKIDPTTGFINAFKRDVVSTNDAKENFLMKED SIQYDIEKGLFKFSFDYKNFATHKLTLAKTKWDVYTNGTRIQNMKVEGHWLSMEVELTTKM KELLDDSHIPYEEGQNILDDLREMKDITTIVNGILEIFWLTVQLRNSRIDNPDYDRIISPVLNKNG EFFDSDEYNSYIDAQKAPLPIDADANGAFCIALKGMYTANQIKENWVEGEKLPADCLKIEHAS WLAFMQGERG (SEQ ID NO: 3287). The EGFR_WT crRNA sequence was Ggagatgttgcttctcttaattcc (SEQ ID NO: 3288), the CCR5 crRNA sequence was Tgcacagggtggaacaagatggat (SEQ ID NO: 3289), and the EGFR_E2 crRNA sequence was Ggagatgtcttgatagcgacggga (SEQ ID NO: 3290). The AsCpf1 handle was Taatttctactcttgtagat (SEQ ID NO: 3291).

Cells were grown in 75T flask using RPMI-1640 (10% fetal bovine serum) until they fill up around 50% of the area, trypsinized, washed with PBS, and finally resuspended in Neon Electroporation Buffer R. 150,000 cells with 500 ng of the Cas12a/crRNA expression were loaded in a 10 μL Neon Pipette tip, and electroporated with an optimized condition (1300V, 20 ms, 2 pulses). Cells were then recovered in the growth media for 6 days and the amount of the cells were measured using luminescence. Compared to the control group, EGFR_WT, for which HCC827 has no corresponding sequence in its genome, the CCR5-targeted sample showed 76% death rate and the EGFR_E2-targeted sample showed 83% death rate. The results showed that the use of Cas12a can induce double- or multi-cleavage derived cancer cell death and the effect can be controlled by customized crRNA designs.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220313796A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. A method of treating cancer of a subject in need thereof, the method comprising: administering to the subject a chimeric polypeptide comprising a first domain comprising a sequence-specific programmable endonuclease and a second domain; and a guide nucleic acid comprising a sequence complementary to a target nucleic acid in a cell of the subject, wherein, upon contacting of the target nucleic acid, the chimeric polypeptide cleaves the target nucleic acid, whereby treating the cancer.
 2. The method of claim 1, wherein the target nucleic acid comprises a cancer-specific sequence.
 3. The method of claim 2, wherein the cancer-specific sequence comprises a single nucleotide polymorphism specific to a cancer, a translocation, a chromosomal abnormality, or a sequence associated with cancer progression.
 4. The method of claim 3, wherein the chromosomal abnormality is selected from the group consisting of a translocation, a deletion, a duplication, an inversion, an insertion, a ring, copy number variations, an indel, and an isochromosome.
 5. The method of claim 1, wherein the target nucleic acid is an oncogene.
 6. The method of claim 1, wherein the target nucleic acid comprises DNA.
 7. The method of claim 1, wherein the target nucleic acid comprises RNA.
 8. The method of claim 1, wherein the target nucleic acid is absent in the healthy cell of the subject.
 9. The method of claim 1, wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of BRCA-1, BRAF, BCR-ABL, HER2, KIF5A, IRX1, ADAMTS16, GNPDA2, KCNE2, SLC15A5, SMIM11, DACH2, HERC2P2, CD68SHBG, ERBB2, KRT16, LINC00536, TRPS1, CDK8, TRAPPC9, HERC2P2, SIRPB1, MRC1, ATP 11 A, POTEB, HERC2P2, PRDM9, CDKN2B, HPV, LINE2 (MT2), CCR5, and HPRT1.
 10. The method of claim 1, wherein the chimeric polypeptide cleaves the target nucleic acid at one or more cleavage sites.
 11. The method of claim 1, wherein the cell is a cancer cell.
 12. The method of claim 1, wherein the cell is a healthy cell.
 13. The method of claim 1, wherein the cancer comprises lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, or cervical cancer.
 14. The method of claim 1, wherein the chimeric polypeptide comprises inducible non-specific nuclease activity.
 15. The method of claim 14, wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide.
 16. The method of claim 1, wherein the first domain comprises a Cas12a (or Cpf1), a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas13a, a Cas13b, a Cas14, or a Cas9.
 17. The method of claim 16, wherein the first domain comprises the Cas9.
 18. The method of claim 1, wherein the second domain comprises a RecE, a RecJ, a T5, a Lambda Exonuclease, a RecBCD, a DNA pol I small fragment, an Exo1, an ExoII, an ExoVII, an Exo T, a Trex1, a mung bean nuclease, or a Trex2.
 19. The method of claim 18, wherein the second domain comprises the RecJ.
 20. The method of claim 1, wherein the second domain comprises an enzyme having cleaved end resection activity. 